CN101401002A

CN101401002A - Nanoporous substrates for analytical methods

Info

Publication number: CN101401002A
Application number: CNA2006800486244A
Authority: CN
Inventors: M·弗拉里; M·M·成; G·库达; M·加斯帕里; D·格欧; L·廖塔; E·彼得里科恩; F·罗伯逊; R·泰拉哈诺
Original assignee: Ohio State University Research Foundation; University of Texas System
Current assignee: Ohio State University Research Foundation; University of Texas System
Priority date: 2005-12-20
Filing date: 2006-12-20
Publication date: 2009-04-01

Abstract

Nanoporous materials can be used to enrich samples for subsequent analysis of substances contained in the sample. The method is shown to enrich the yield of species in the low molecular weight proteome, allowing detection of small peptides in the low nanomolar range.

Description

Be used for the nanoporous substrate of analytical approach

Priority request

[0001] the application requires the right of priority of the U.S. Provisional Patent Application submitted on Dec 20th, 2005 number 60/751,924, its complete being incorporated herein by reference here.The application also requires on Dec 15th, 2006 to submit to, the right of priority that the U.S. Provisional Patent Application of Mauro Ferrari etc. " is used for the nanoporous substrate (Nanoporous Substrates for the Analysis ofBiological Fluids) that biofluid is analyzed ", it is this complete being incorporated herein by reference.

Federal funds are subsidized the research statement

[0002] researchs more of the present invention are by the national ICR (National Cancer Institute) from national health research institute (NationalInstitutes of Health), and contract number is that the Federal funds of NO1-CO-12400 are supported.Government enjoys some right of the present invention.

Invention field

[0003] the application's relate generally to analytical equipment and system and production and preparation method thereof more particularly, relate to the analytical equipment of using nano-porous materials and system and production and preparation method thereof.

Background of invention

[0004] technology of material in the multiple available analysis clinical sample is arranged, but most of common technologies requires expensive solution to solve the interference that material highly abundant in the sample produces, as the albumin in the serum situation.A solution of this problem comprises the material that uses the antibody capture height abundant, and lowers its existence in sample, so that it does not disturb the analysis to other desired substance.Existence is to the demand of better method and system, so that the material in the analytic sample.

Summary of the invention

[0005] in one embodiment, the invention provides the method for a kind of classification or separation, comprising: a kind of sample that contains first component and second component (a) is provided; (b) provide a kind of substrate that contains nano-porous materials; (c) described nano-porous materials is exposed to sample, wherein when exposing, described nano-porous materials keeps described first component but does not keep described second component.

[0006] in another embodiment, the invention provides the method for analytic sample, comprising: (a) sampling; (b) provide a kind of substrate that contains nano-porous materials; (c) described nano-porous materials is exposed to sample; And analyze sample part by nano-porous materials kept.

[0007] and in other a embodiment, the invention provides a kind of method that detects the physiological condition mark, comprising: a kind of sample that is influenced by physiological condition (a) is provided; (b) provide a kind of substrate that contains nano-porous materials; (c) described nano-porous materials is exposed to sample; (d) analyze the sample part that is kept by nano-porous materials; (e) analysis result with income analysis result and control sample compares, to detect the mark of physiological condition.

[0008] in another embodiment, the invention provides a kind of kit, it comprises the apparatus of collecting the sample that comprises one or more components; With the substrate that comprises the nano-porous materials that is configured to keep one or more components.

[0009] and in another embodiment, the invention provides a kind of analytic system, it comprises analytical instrument and a kind of substrate that comprises nano-porous materials, and wherein said substrate has been configured to strengthen the susceptibility of described analytical instrument to one or more analytes.

[0010] and in another embodiment, the invention provides a kind of probe that comprises substrate, described substrate comprises a kind of nano-porous materials and is configured to and can insert in the mass spectrometer.

Accompanying drawing

[0011] Fig. 1 has shown scanning electron microscope (ScanningElectron Microscopy (the SEM)) image of silicon dioxide A and B.

[0012] Fig. 2 (A)-(C) has shown human serum dilute sample (Fig. 2 A); Be exposed to the human albumin (Fig. 2 B) who keeps behind the A type nano-stephanoporate silicon dioxide particle; With (Matrix Assisted Laser Desorption Ionization-Time OfFlight (the MALDI-TOF)) mass spectrogram of substance assistant laser desorpted ionized flight time that is exposed to the human albumin (Fig. 2 C) who keeps behind the Type B nano-stephanoporate silicon dioxide particle.

[0013] Fig. 3 (A)-(C) has shown (MALDI-TOF) mass spectrogram of substance assistant laser desorpted ionized flight time that is exposed to the human albumin who keeps behind the A type nano-stephanoporate silicon dioxide particle.A, B, C represent one group of three independent experiment.

[0014] Fig. 4 (A)-(C) has shown the substance assistant laser desorpted ionized flight time mass spectrum figure that is exposed to the human albumin who keeps behind the Type B nano-stephanoporate silicon dioxide particle.A, B, C represent one group of three independent experiment.

[0015] Fig. 5 (a)-(d) has shown the mass spectrogram of the doping experiment (spiking experiment) of insulin under four variable concentrations: a) 500ng/mL; B) 200ng/mL; C) 30ng/mL; And d) 15ng/mL.

[0016] Fig. 6 has schematically shown: use the nanoporous Silicon Wafer, the strategy of part dilution serum (partial depletion of serum).The nanoporous Silicon Wafer is produced, and bathes with the serum temperature.Temperature is bathed after date, described wafer is removed, and remaining blood serum sample point sample is carried out surface-enhanced laser desorption ionization (SurfaceEnhanced Laser Desorption Ionization (SELDI)) mass spectrum (MS) analyze on the weak cation exchange chip, and with the mass spectrogram of the control group serum that is not exposed to the nanoporous wafer relatively.

[0017] Fig. 7 has shown the result of the mass spectrophotometry of using nano-structure porous silicon wafer segment consumption serum.As describing among Fig. 6, the nanoporous Silicon Wafer of serum and aminopropyl group bag quilt together temperature is bathed.After temperature was bathed, described serum was incorporated on the weak cation exchange chip, and carries out mass spectrophotometry subsequently.Obtained mass spectrum (MS) fingerprint.Natural sera (not being exposed to the serum of nanoporous wafer) compares with the serum that consumes through wafer.A main peak (arrow on redness/the right) appears in the natural sera, and it has covered a less peak (arrow on blueness/left side) in similar m/z scope.When bathing, partly consume in the serum of its protein group composition, compare, become main peak by the peak of peak and the redness/right side arrow institute mark of blueness/left side arrow indication with the temperature of nanoporous wafer.When the ratio of more relative peak intensity between the peak on blueness/left side and redness/right side, demonstrate significant skew at natural sera with between with the serum after the consumption of nanoporous Silicon Wafer.

[0018] Fig. 8 has schematically shown the strategy of collecting molecule with the nanoporous beaded glass from serum.Beaded glass and serum temperature with aminopropyl group bag quilt are bathed.Temperature is bathed after date, removes pearl, cleans, and with the molecule wash-out of combination, and point sample carries out SELDI to the weak cation exchange chip to be analyzed, and with the mass spectrogram of the control serum that is not exposed to the nanoporous pearl relatively.

[0019] Fig. 9 has shown the mass spectrometry results of the molecule of being collected by the nanoporous beaded glass.Describe as Fig. 8, beaded glass one equality of temperature of serum and aminopropyl group bag quilt is bathed.After temperature was bathed, described serum at first was attached on the weak cation exchange chip, and carries out mass spectrophotometry subsequently.The molecular proportion that natural sera (not being exposed to the serum of nanoporous pearl) and usefulness 17nm pearl are collected.In natural sera, main peak (arrow on redness/the right) has covered a less peak (arrow on blueness/left side) in similar m/z scope.In the sample of wash-out, promptly be exposed in the sample of nanoporous beaded glass, compare by the peak of peak and the redness/right side arrow institute mark of blueness/left side arrow indication, become main peak.The ratio of the peak-to-peak relative peak intensity on blueness/left side and redness/right side is at natural sera with collect and between the molecule of wash-out, demonstrate significant skew from the nanoporous pearl subsequently.

[0020] Figure 10 has shown the mass spectrometry results of the molecule of collecting with 70nm hole pearl.Describe as Fig. 8, serum with have the 70nm hole and bathed by beaded glass one equality of temperature of aminopropyl group bag quilt.After temperature was bathed, serum was attached on the weak cation exchange chip, and carries out mass spectrophotometry subsequently.The molecular proportion that natural sera (not being exposed to the serum of the brilliant pearl of nanoporous) and usefulness 70nm pearl are collected.In similar m/z scope, main peak (arrow on redness/the right) has covered a less peak (arrow on blueness/left side).In the sample of wash-out, the peak of blue arrow indication is compared with the peak of red arrow institute mark, becomes main peak.The ratio of the peak-to-peak relative peak intensity on blueness/left side and redness/right side is at natural sera with collect and demonstrate significant skew between the molecule of wash-out from this pearl subsequently.

[0021] Figure 11 has shown the natural sera that is not exposed to nano-porous materials and has been the result that the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sodium Dodecyl Sulfate-Polyacryl Amide GelEletrophoresis (SDS-PAGE)) of molecule of the beaded glass wash-out of the beaded glass of 17nm and aperture 70nm is analyzed from the aperture.

[0022] Figure 12 shows the peptide sequencing result, and it has shown the protein sequence that obtains from the eluate of the beaded glass of the beaded glass of aperture 17nm and aperture 70nm.

[0023] Figure 13 (A)-(C) has compared use nano-porous materials (A) and classical tissue chemical technology (B, C) serology classification and the purifying enhancing to disclose pathology identification body (discriminator).Figure 13 (A) has shown that serum is exposed to the detection that the nanoporous surface has promoted some protein peak.Figure 13 (B): the trichrome stain of Masson (right side drawing) has shown collagen deposition, and this is to carry out the hepatic fibrosis-renal tubular ectasia syndrome relevant with chronic hepatitis C infection necessary information (left side drawing, conventional H stage by stage for the patient; E dyeing).

[0024] Figure 13 (C): Bielchowsky silver impregnation method has disclosed the neuritic plaques (neuritic plaque) (right side drawing) of alzheimer disease cerebral neocortex tissue, can not be by H﹠amp; The main pathology sign (left side drawing) that E dyeing detects.

[0025] Figure 14 has shown the form of the silica nanometer perforated membrane of use transmission electron microscope (Transmission ElectronMicroscopy (TEM)), the embodiment 3 that details vide infra.

[0026] Figure 15 (i)-has (ii) shown the collection of illustrative plates of the MALDI-TOF of the human plasma after temperature is bathed on the particular surface.(i) with the blood plasma of nano-stephanoporate silicon dioxide chip temperature bath; The (ii) blood plasma of bathing with solid-state silica chip temperature.The sample of analyzing is the human plasma that is added with 5 microlitre equal portions of 1mg/mL concentration calcitonin.The peak of described calcitonin asterisk ( ^*) mark.

[0027] Figure 16 has shown by using the nano-stephanoporate silicon dioxide chip to collect the repeatability of the MALDI-TOF peptide mapping that obtains.

[0028] Figure 17 (i)-has (iv) shown that blending has the low-molecular-weight (low molecular weight (LMW)) of the blood plasma of people's calcitonin to collect.Four experiments (in the m/z window around the calcitonin peak, changing) have been shown: (i) 1000ng/mL, (ii) 200ng/mL, (iii) 50ng/mL, (iv) 20ng/mL with the calcitonin doped in concentrations profiled blood plasma that reduces.Temperature bathe and the mass spectrum condition as described in the embodiment 3 of experimental section hereinafter.

[0029] Figure 18 has schematically shown the strategy of a kind of carrier of design (ship), and this carrier is used for strengthening the susceptibility of the low molecular weight protein group (lowmolecular weight proteome (LMWP)) that embodiment 4 hereinafter goes through.

[0030] Figure 19 has shown the MALDI-TOF mass spectrogram of three kinds of different sample/matrix MALDI prepared products.

[0031] Figure 20 has shown the MALDI-TOF mass spectrogram of control group mice serum.

[0032] Figure 21 has shown the MALDI-TOF mass spectrogram of the mouse control serum that uses four kinds of different nanoporous pearls.Upper left drawing: the silica nanometer porous pearl of small-bore; Upper right drawing: wide-aperture silica nanometer porous pearl; Lower-left drawing: with the small-bore silica nanometer porous pearl of 3-sulfydryl propyl trimethoxy silicane (3-mercaptopropyltrimethoxysilane (MPTMS)) modification; Bottom right drawing: with the large aperture silica nanometer porous pearl of 3-aminopropyltriethoxywerene werene (3-aminopropyltrimethoxysilane (APTES)) modification.

[0033] Figure 22 shows the 1D gel electrophoresis result of small-bore silicon dioxide pearl eluate sample.The Tris-glycocoll gradient gel (8-16% acrylamide) of the pearl eluate of acquisition from the combining anteserum (pooled serum) of bathing with small-bore silicon dioxide pearl temperature.The corresponding molecular weight standard of the swimming lane of high order end, and remaining swimming lane is from left to right sequentially corresponding to

pearl eluate sample

1,7,13,19,25,31,37,43,49,55,61 and 67 (key word of sample sign, the table 4 among the embodiment 5 that vide infra).

[0034] Figure 23 shows the 1D gel electrophoresis result of large aperture silicon dioxide pearl eluate sample.The Tris-glycocoll gradient gel (8-16% acrylamide) of the pearl eluate that from the combining anteserum of bathing with large aperture silicon dioxide pearl temperature, obtains.The corresponding molecular weight standard of the swimming lane of high order end, and the remaining from left to right sequentially corresponding

pearl eluate sample

4,10,16,22,28,34,40,46,52,58,64 of swimming lane and 70 (key word of sample sign vide infra the table 4 among the embodiment 5).

[0035] Figure 24 shows the 1D gel electrophoresis result of the silicon dioxide pearl eluate sample of small-bore APTES modification.The Tris-glycocoll gradient gel (8-16% acrylamide) of the pearl eluate that from the combining anteserum of the pearl temperature bath of modifying with small-bore APTES, obtains.The corresponding molecular weight standard of the swimming lane of high order end, and the remaining from left to right sequentially corresponding

pearl eluate sample

2,8,14,20,26,32,38,44,50,56,62 of swimming lane and 68 (key word of sample sign vide infra the table 4 among the embodiment 5).

[0036] Figure 25 shows the 1D gel electrophoresis result of the silicon dioxide pearl eluate sample of large aperture APTES modification.The Tris-glycocoll gradient gel (8-16% acrylamide) of the pearl eluate that from the combining anteserum of the pearl temperature bath of modifying with large aperture APTES, obtains.The corresponding molecular weight standard of the swimming lane of high order end, and the remaining from left to right sequentially corresponding

pearl eluate sample

5,11,17,23,29,35,41,47,53,59,65 of swimming lane and 71 (key word of sample sign vide infra the table 4 among the embodiment 5).

[0037] Figure 26 shows the 1D gel electrophoresis result of the silicon dioxide pearl eluate sample of small-bore MPTMS modification.The Tris-glycocoll gradient gel (8-16% acrylamide) of the pearl eluate that from the combining anteserum of the pearl temperature bath of modifying with small-bore MPTMS, obtains.The corresponding molecular weight standard of the swimming lane of high order end, and the remaining from left to right sequentially corresponding

pearl eluate sample

3,9,15,21,27,33,39,45,51,57,63 of swimming lane and 69 (key word of sample sign vide infra the table 4 among the embodiment 5).

[0038] Figure 27 shows the 1D gel electrophoresis result of the silicon dioxide pearl eluate sample of large aperture MPTMS modification.The Tris-glycocoll gradient gel (8-16% acrylamide) of the pearl eluate that from the combining anteserum of the pearl temperature bath of modifying with large aperture MPTMS, obtains.The corresponding molecular weight standard of the swimming lane of high order end, and the remaining from left to right sequentially corresponding

pearl eluate sample

6,12,18,24,30,36,42,48,54,60,66 of swimming lane and 72 (key word of sample sign vide infra the table 4 among the embodiment 5).

[0039] Figure 28 has shown that protein band cuts out collection of illustrative plates in the gel of Figure 27.Gel images is the copy of Figure 27 gel, has the subsides notes (overlay) of the band excision pattern that is used to obtain the tandem mass spectrum specimen in use.From every swimming lane, excise out the low-molecular-weight band, and all protein bands in the 8th swimming lane are cut out.The numbering of sample and sign key word are as follows.Band in every swimming lane is cut out from top to bottom, from the 2nd road (the 1st road is corresponding to molecular weight standard) in left side and proceed to the 7th road.Skip the 8th road, continue to cut out bar and take the 13rd road to from the 9th road.Each bar from these swimming lanes cuts out 4 bands.Since the 2nd swimming lane, the band on top is corresponding to the A1 hole, to the band of bottom, corresponding to the D1 hole.Afterwards, cut out band from the 8th swimming lane, from the band on top, the E6 hole begins, to the band G8 of bottom.The 2nd swimming lane, sample well A1-D1; The 3rd swimming lane, sample well E1-H1; The 4th swimming lane, sample well A2-D2; The 5th swimming lane, sample well E2-H2; The 6th swimming lane, sample well A3-D3; The 7th swimming lane, sample well E3-H3; The 9th swimming lane, sample well A4-D4; The 10th swimming lane, sample well E4-H4; The 11st swimming lane, sample well A5-D5; The 12nd swimming lane, sample well E5-H5; The 13rd swimming lane, sample well A6-D6; The 7th swimming lane, sample well E6-G8.

[0040] Figure 29 shows the collection of illustrative plates that protein band cuts out among Figure 25.The gel image is the low-molecular-weight zone of gel among Figure 25, and the 10th swimming lane has the subsides note that is used for from the band excision pattern of 12 swimming lanes and 13 swimming lanes acquisition tandem mass spectrum specimen in use to the copy of 13 swimming lanes.From 12 swimming lanes and 13 swimming lanes, cut out the low-molecular-weight band.Sample number into spectrum and sign key word are as follows.From the 12nd swimming lane on the left side, and proceed to 13 swimming lanes, cut out the band in every swimming lane from top to bottom.Each bar from these swimming lanes cuts out 5 bands.Since the 12nd swimming lane, the band counter sample hole H8 on top is to the bottom band corresponding to sample well D9.Cut out band from the 13rd swimming lane, from the top band, hole E9 begins, to low side strips A 10.

[0041] Figure 30 shows the protein band excision collection of illustrative plates of gel among Figure 26.Gel images is the copy of the gel of Figure 26, has the subsides note of the band excision pattern that is used to obtain the tandem mass spectrum specimen in use.Only from the 6th swimming lane, cut out all protein bands.Sample number into spectrum and sign key word are as follows.From up to down cut out band from the 6th swimming lane.From this swimming lane, cut out 17 bands altogether.From the band on top, this sample is corresponding to hole B10, to the bottom band corresponding to hole B12.The band that begins from the gel top is corresponding to sample well B10, C10, D10, E10, F10, G10, H10, A11, B11, C11, D11, E11, F11, G11, H11, A12 and last B12.

[0042] Figure 31 shows the SELDI mass spectrogram of combining anteserum.Shown from the SELDI spectrum that merges and the WCX2 chip of the former serum of dilution obtains.Each sample is characterized by duplicate collection of illustrative plates.From the top: collection of

illustrative plates

1 and 2, the 28 days-clone's 8 serum; Collection of

illustrative plates

3 and 4, the 28 days-clone 10; Collection of illustrative plates 5 and 6, the 42 days-clone 8; And collection of illustrative plates 7 and 8, the 42 days-clone 10.

[0043] Figure 32 shows the SELDI mass spectrogram of combining anteserum.Shown from the SELDI spectrum that merges and the WCX2 chip of the former serum of dilution obtains.Each sample is characterized by duplicate collection of illustrative plates.From the top: collection of

illustrative plates

1 and 2, the 60 days-matrigel (matrigel) control serum; Collection of

illustrative plates

3 and 4, the 60 days-BT474 contrast; Collection of illustrative plates 5 and 6, the 60 days-MCF7 control cells system; And collection of illustrative plates 7 and 8, the 60 days-clone 8.

[0044] Figure 33 shows the SELDI mass spectrogram of combining anteserum.Shown from the SELDI spectrum that merges and the WCX2 chip of the former serum of dilution obtains.Each sample is characterized by duplicate collection of illustrative plates.From the top: collection of

illustrative plates

1 and 2, the 60 days-clone's 10 serum; Collection of

illustrative plates

3 and 4 merges control group A; Collection of illustrative plates 5 and 6 merges control group B; And collection of illustrative plates 7 and 8, merge control group C.

[0045] Figure 34 (A)-(C) shows the SELDI spectrum of pearl eluate.

[0046] Figure 35 (A)-(C) shows the SELDI spectrum of pearl eluate.

[0047] Figure 36 (A)-(C) shows the SELDI spectrum of pearl eluate.

[0048] Figure 37 (A)-(C) shows the SELDI spectrum of pearl eluate.

[0049] Figure 38 (A)-(C) shows the SELDI spectrum of pearl eluate.

[0050] Figure 39 (A)-(C) shows the SELDI spectrum of pearl eluate.

Detailed Description Of The Invention

[0051] except as otherwise noted, word "/(" a ") or/" an ") " is with representing herein " one or more ".

[0052] in one embodiment, the invention provides a kind of method, it comprises provides the sample that contains the first component and second component, and the substrate that contains nano-porous materials is provided, and described nano-porous materials is exposed to described sample. During exposure, described nano-porous materials keeps described the first component but does not keep described second component.

[0053] preferably, described sample is biological sample,, contains the sample of biomolecule, such as albumen, peptide, antigen, antibody, protein fragments, RNA or DNA that is. Described biological sample can be from plant; Animal comprises mammal, and is preferably human; Or the sample of cell culture. Described biological sample can be the sample of biofluid, such as blood (blood), serum (blood serum), blood plasma (blood plasma), urine (urine), seminal fluid (seminal fluid), refining (seminal plasma), liquor pleurae (pleural fluid), ascites (ascites), milk (nipple aspirate), ight soil (feces) or saliva (saliva).

[0054] described nano-porous materials can be pore-size distribution centered by less than 1000nm, preferably less than any material centered by the 100nm. In some embodiments, described nano-porous materials can be nano-structure porous silicon. And in other embodiments, described nano-porous materials can be the nanoporous oxidation material, such as nano-stephanoporate silicon dioxide or nano-porous alumina.

[0055] described nano-porous materials can separate the first component and second component by molecular weight, also, by the first component that nano-porous materials keeps, compares with second component, has less mean molecule quantity.

[0056] component that is kept by nano-porous materials can be adsorbed onto nano-porous materials, that is, it is the component that can be washed away by gentleness, such as the component of going with the deionized water washing.

[0057] first component that is kept by nano-porous materials can be a lower-molecular-weight component, that is, a kind of component, wherein the molecular weight of nearly all molecule is not higher than 20kDa, or is not higher than 15kDa, or is not higher than 10kDa, or is not higher than 5kDa or is not higher than 4kDa.

[0058] described nano-porous materials can be brought into play the effect that molecule is held back (cut off), be that described nano-porous materials can keep molecular weight and is equal to or less than molecule and holds back all of weight or all molecules basically, but do not keep molecular weight is held back weight greater than molecule all or nearly all molecule.The molecule of described nano-porous materials is held back weight and can be changed by the aperture of adjusting this nano material.

[0059] surface of described nano material can be modified, for example, and by being accumulated in this lip-deep electric charge or modified with functional group.Such modification can be used to the specific components of keeping sample.For example, can as amino silane, modify described surface by with containing amino molecule so that positive charge to be provided.Can as hydrosulphonyl silane, modify described surface by with the molecule that contains mercapto groups so that negative charge to be provided.Chain alkyl also can be contained (greater than C by accumulation in described surface ₁₀) molecule, as alkyl silane, modify with hydrophobic grouping.

[0060] the also available metal such as copper or iron in the surface of described nano-porous materials is modified, and this can improve this nano-porous materials to the sample specific components---as phosphorylated protein---affinity.Modify nanoporous oxide surface, can in the building-up process of this nanoporous oxide, add the salt of described metal such as nano-stephanoporate silicon dioxide.For example, for nano-stephanoporate silicon dioxide, the Cu (MeCO of different amounts ₂) 2H ₂O can be added into CTAB (hexadecyl trimethyl ammonium bromide (cetyltrimethylammonium bromide)) and TEOS (tetraethyl orthosilicate (tetraethylorthosilicate)) and Na ₂O and H ₂Among the O.Afterwards, can carry out pyroprocessing at about 200 degrees centigrade and about 540 degrees centigrade.The surface of nano-structure porous silicon can be modified with metal by using chemical deposit (electroless plating).

[0061] substrate that contains nano-porous materials can provide with various forms, includes but not limited to film, wafer, particle or microchip.

[0062] in some embodiments, described substrate can be with top-down technology manufacturing, as photoetching process, el, X ray etching, deep-UV lithography and nano impression method.

[0063] in some embodiments, the component that is kept by nano-porous materials in the sample can be extracted from sample, for example to carry out further analysis or to observe.

[0064] in other embodiments, can under the situation of not extracting first component, further analyze or observe the sample component of nano-porous materials reservation.

[0065] can pass through the example gel electrophoresis to the further analysis of first component, as SDS-PAGE; Chromatogram; Bioassay technique or mass spectrum carry out as MALDI-TOF mass spectrum, LS/MS mass spectrum, electrospray ionization mass spectrum, tandem mass spectrum or SELDI mass spectrum.

[0066] before analysis, described sample is exposed to the detection level that described nano-porous materials can strengthen this analysis.For example, sample is exposed to nano-porous materials, mass spectrum can not be higher than 1000ng/mL by detectable concentration, or be not higher than 200ng/mL, be not higher than 100ng/mL, or be not higher than 20ng/mL, or be not higher than 10ng/mL, or be not higher than 5ng/mL, or be not higher than the existence of the low-molecular-weight molecule of 1ng/mL.

[0067] substrate that contains described nano-porous materials also can be used for detecting and/or discerning the biomarker of physiological condition, as the biomarker in stage of disease or disease.Disease can be, cancer for example is as breast cancer.

[0068] to detect and/or discern the biomarker of physiological condition, we can be exposed to the sample that is influenced by this physiological condition the substrate that contains nano-porous materials, analyze the part of the sample of nano-porous materials reservation, and the result that will analyze---as mass-spectrogram, compare the sample that control group is not promptly influenced by this physiological condition to the similar analysis result of control group.

[0069] substrate that contains described nano-porous materials also can be used to collect and/or store biological sample.For example, in some embodiments, the substrate that contains nano-porous materials can be the part of kit, and this kit also comprises the feasible instrument of any collection of biological sample.The sample of collecting can be exposed to described nano-porous materials, and stores subsequently, for use in later analysis or observation.

[0070] in one embodiment, the substrate that contains described nano-porous materials can be based on the part of the analytic system of particular analysis instrument.Under these circumstances, described substrate can specifically be configured to strengthen analytical instrument to one or more analytes, as the susceptibility of low-molecular-weight biomolecule.

[0071] for such application, described substrate can have one or more zones of containing described nano-porous materials.The zone that the goal analysis thing is not adsorbed in each such zone surrounds.Enclosing region is non-nano porous zone preferably, and promptly it does not contain nano-porous materials.The available functional group that absorption goal analysis thing is had resistance makes enclosing region passivation (passified).Under the situation of peptide and albumen, described enclosing region can be used hydrophilic functional groups, as the polymkeric substance that contains PEG is modified.

[0072] in some embodiments, described substrate can be with in the sample sets to be analyzed or be concentrated in one or more zones of containing described nano-porous materials.Concentrating or concentrating like this can reduce the sample size that is exposed to analysis, and this can strengthen the susceptibility to the goal analysis thing again.

[0073] in some embodiments, the size that contains the zone of described nano-porous materials can be made into to meet the size of the active region of described analytical instrument ionization source.For example, when described analytical instrument was analyzed the ionization of laser, the area size that contains described nano-porous materials can meet the size (diameter) of laser beam.Such substrate is particularly useful to using laser to carry out Ionized mass spectrometer, as MALDI mass spectrometer or SELDI mass spectrometer.

[0074] in some embodiments, the substrate that contains nano-porous materials can be configured to insert mass spectrometric probe, as MALDI mass spectrometer or the mass spectrometric probe of SELDI.

[0075] the present invention is further by following examples, but illustrates in unrestriced mode.

Embodiment 1

[0076] quoting in the present embodiment bracket refers to embodiment 1 last list of references and tabulates.

1. brief introduction

[0077] Proteomic analysis of the human plasma/serum of cancer and other disease early detection concerning many research groups, is an interested day by day field [1,2 and list of references wherein].More concern concentrates on the low-molecular-weight molecule that combines with carrier protein especially, and it is considered to produce and constitutes and contains the most of ion [3-5] that is useful on unique MS collection of illustrative plates of finding biomarker.Therefore, be designed to imitate carrier protein (as albumin) in conjunction with and carry out the design and development of the particle that low molecular weight protein (LMWP) group (low molecular weight proteome (LMWP)) collects, the discovery of biomarker is had a significant impact [6-8].Based on the mass spectral protein chip array system from Ciphergen of SELDI-TOF (ProteinChip Array System) is the most popular platform [9,10] of finding " molecular signal (molecular signature) ".In the nearest technical progress in this field, the promising method of appearance comprises: new peptide group (peptidomics) platform, and its magnetic base with little peptide, automatic solid phase extractions and MALDI-TOF mass spectrum reading associate [11,12]; In the modification of surfaces desorb/ionization (DIOS) [13] of silicon and the desorb/ionization [14] on the silicon nanowires (Silicon Nanowires (SiNWs)).For the discovery based on the biomarker of protein group, nanometer technology can offer an opportunity and challenge [15].The potentiality of nanometer technology to be transformed on the proteomics, study nano-stephanoporate silicon dioxide and be applied to screen plasma proteins, its objective is more effective and collect blood plasma LMWP efficiently.

[0078], carried out the substance assistant laser desorpted ionized flight time (matrix assisted laser desorptionionization-time of flight (MALDI-TOF)) mass spectrophotometry to plasma proteins from two kinds of different nano-stephanoporate silicon dioxide surface extraction.The mass spectrogram of gained has shown that the nanometer grade silica particle keeps the ability of up to a hundred peptides and small molecular weight protein.

2. material and method

2.1 material and instrument

[0079] the synthetic reagent of gel comprises sodium silicate solution (Sigma, St Louis, MO, the U.S.), aerosil (fumed silica) (Sigma-Aldrich, St.Louis, MO, the U.S.), (Nonfix 10, Condea, Houston for the different nonylplenyl ether of polyoxyethylene (10), TX, the U.S.) and hexadecyl trimethyl ammonium bromide (CTABr, Aldrich, St.Louis, MO, the U.S.).MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) obtains from Sigma company (St.Louis, MO, the U.S.).Protease inhibitors cocktail (PIC: heparin lithium (lithiumheparin), EDTA, AEBSF, bestatin (bestatin), E-64, leupeptin (leupeptin) and Aprotinin (aprotinin)) is from (the St.Louis of Sigma company, MO, the U.S.) buy.Pancreas Bovis seu Bubali insulin obtains from Sigma company (St.Louis, MO, the U.S.).

[0080] all samples are at Applied Biosystems Voyager-DE ^TMThe light of the 337nm that (Framingham, MA, the U.S.) sends with nitrogen laser on the STR mass spectrometer is analyzed.Analysis is carried out with linear model.

2.2 synthesis step

[0081] silica sample A obtains according to two kinds of different route of synthesis with B, described in [16,17].Briefly, silica sample A and B rise at first with the gel of the component that adds following molal quantity and obtain: silicon dioxide A SiO ₂: 0.064 Nonfix 10,0.6NaOH, 0.8 HCl, 58 H ₂O; Silicon dioxide BSiO ₂: 0.2 CTABr, 0.2 NaOH, 0.04Al (OH) ₃, 40 H ₂O.

[0082] to silicon dioxide A, (2.9g Nonfix 10 and 4.05gNaOH are dissolved in 57.4g H at surfactant solution ₂Among the O) disperse fully after, add the 14.6g sodium silicate.At last, add the HCl of 5.33g37%wt%, and this gel stablized at room temperature 24 hours, 100 ℃ of heating 24 hours in baking box subsequently.

[0083] silicon dioxide B is by to containing 0.52g Al (OH) ₃, 1.35g NaOH and 130g H ₂Adding the 10g aerosil in the solution of O obtains.Described gel was at room temperature stablized 2 hours, heated 24 hours in 140 ℃ baking box subsequently.Described synthesized gel rubber is filtered, and uses deionized water rinsing, and 80 ℃ of dryings 12 hours.

[0084] the nitrogen adsorption desorb volume isotherm under 77K is measured on MicrometriticsAsap 2010 equipment.Sample 300 ℃ of bakings in a vacuum spends the night, and benzoylated (benziylated) sample is handled identical surplus pressure at 230 ℃.The surface area of described sample is by arriving 0.2p/P 0.005 ₀Pressure limit in Brunauer-Emmet and Teller (BET) linearization [16] that obtain.

2.3 plasma collection

[0085] human plasma obtains according to the guidance of suggestion in [18].Simply, blood is collected into and adds the 8mL heparin lithium separating plasma pipe (PST that 300 μ L PIC are arranged in advance ^TM

367965, Becton Dickinson, Franklin Lakes, NJ, the U.S.) in, and in 15 minutes of blood drawing, under 4 ℃, centrifugal 15 minutes of 2500g.In the centrifugal end 30 minutes, plasma layer is divided into the equal portions of 1.0mL; And it is freezing rapidly with dry ice/ethanol bath.

2.4 experimental procedure

[0086] sample aliquot of silica dioxide granule (5mg) is mixed with 500 μ L human plasma dilute samples (1:5), and at room temperature shakes 1 hour.Suspension centrifugal 2 minutes at 2000xg, silica dioxide granule is separated from supernatant and with deionized water wash (4 x, 100 μ L) subsequently.The plasma proteins that silica surface keeps extracts as follows: silica dioxide granule is suspended in 100 μ L solution (in water/methyl alcohol of 4.95:4.95:0.1/0.1%TFA), and with 2000xg centrifugal 5 minutes rapidly.Contain the supernatant that extracts albumen, analyze with MALDI-TOF-MS.1 μ L supernatant mixes with 4 μ LCHCA matrix, and 1 μ L gained solution is also air-dry in the MALDI dish by point sample subsequently.

The experiment 2.5 mix

[0087] plasma sample mixes with the insulin of four kinds of variable concentrations: 500ng/mL, 200ng/mL, 30ng/mL and 15ng/mL.Described sample is exposed to silicon dioxide A afterwards as described in 2.3 parts.At last, 5 μ L blood plasma extract albumen and are advanced in the 3 μ LCHCA matrix by wash-out, and 1 μ L gained solution is also air-dry in the MALDI dish by point sample subsequently.

3-4 result and discussion

[0088] in this research, studied that novel nano porous silica particle is caught and the ability of enrichment low molecular weight peptide and albumen from human plasma.These archives are considered to contain the undeveloped resources [3-5] at biomarker potential, that disease is special now.This news file is filed rapidly and abundant effective way, can appreciable impact be arranged the discovery of biomarker.

[0089] Fig. 1 has shown the form of silicon dioxide A and B.Nitrogen adsorption-desorption isotherm shows that the surface area of silicon dioxide A is 406m ²/ g and pore volume are 0.3cm ³/ g.Be applied to isotherm desorb branch Barret-Joyner-Halenda (BJH) models show the bimodality (bimodality) of hole system, show that the aperture is with 26.8 Hes

Be central distribution.For silicon dioxide B, surface area is 848m ²/ g and pore volume are 1.21cm ³/ g.The BJH pore diameter distribution is with 25 Hes

Be the center.

[0090] investigative test is devised, estimating nano-stephanoporate silicon dioxide to plasma proteins, and especially to the capture ability of low molecular weight protein histone.

[0091] before the analysis of carrying out based on MS, a quick and simple method of sample pre-concentration is developed, it allows the potential degraded of biological sample is reduced to minimum.Compare with untreated sample, the human plasma sample's who handles with silica dioxide granule mass spectrum demonstrates the obvious visible enrichment of LMWP, and this relies on employed specific nano-stephanoporate silicon dioxide substrate, sees Fig. 2.

[0092] two MALDI-TOF spectrum with the sample that nano-stephanoporate silicon dioxide is handled has all shown up to a hundred peaks, particularly in 800 to 5000 daltonian low-molecular-weight intervals.To each batch silicon dioxide, experiment is to carry out in triplicate, to estimate the repeatability of this test.Data show that described method produces repeatably total spectrogram of high-intensity signal (seeing Fig. 3 A-B-C and Fig. 4 A-B-C).High-quality spectrogram may be the product of clear program.Clean silicon dioxide substrate (before the albumen that extraction is caught) and can remove the salt that causes serious signal suppressing potentially, nonvolatile and hydrophobic pollutant.Produce silica dioxide granule effectively and keep the ability (Fig. 3 and 4) of repeated spectral line of molecule also owing to the intrinsic chemical stability of silicon dioxide substrate [16,17].

[0093] insulin (molecular weight 5733.5Da) with variable concentrations carries out the doping of plasma sample, with the test limits (limit of detection (LOD)) of setting up this paper describing method.Concentration is low to moderate the MALDI-TOF signal of the insulin of 15ng/mL in the human plasma, is detected (Fig. 5).

[0094] with this method of MALDI-TOF technical tie-up,, be enough sensitivities to detecting the plasma peptides of every milliliter low nanogram scope.Consider at present as the α subunit of hoptoglobin, to be 1000nmol/L[22,23 with the discernible least concentration of MS to biomarker], the LMWP blood plasma enrichment method of this paper has reduced about 400 times with LOD.

[0095] based on the MS spectrogram, have each in two kinds of silicon dioxide substrates in different apertures, with after identical plasma sample temperature is bathed, kept different peptide/protein combination (repertoire) (see figure 2).Although the present invention is not limit by its theory of operation, can suppose viewed be not both based on the surface properties of substrate and by electrostatic interaction with protein adsorption to the tendency of water-wetted surface.The LMWP combination can be by additional chemistry and structural modification, and the surface properties that makes the silicon dioxide substrate is suitable and strengthen.

5. concluding comment

[0096] demonstrate: the nano-stephanoporate silicon dioxide particle can be by successfully as the entity of similar substrates albumen, and in plasma or other biological fluid the enrichment low-molecular-weight molecule.The result has shown the molecular recognition body attribute of the nano-stephanoporate silicon dioxide particle of the potential selectivity of molecule and selective binding LMWP.Therefore, these new substrates and mass spectrum surface combination, the method that can provide biomarker component self to check order and predict the potential platform of medicine based on LMWP fast.

[0097] 6. list of references tabulation

[1]Anderson，N.L.，Mol.Cell.Proteomics 2005，Oct 2005；4：1441-1444.

[2]Xiao，Z.，Prieto D.，Conrads，T.P.，Veenstra，T.D.，Issaq，H.J.，Mol.Cell.Endocrinol.2005，230，95-106.

[3]Liotta，L.A.，Ferrari，M.，Petricoin，E.，Nature 2003，425，905.

[4]Mehta，A.I.，Ross，S.，Lowenthal，M.，Fusaro et al.，Dis.Markers 2003，19，1.

[5]Tirumalai，R.S.，Chan，K.C.，Prieto，D.A.，Issaq，H.J.et al.，Mol.Cell.Proteomics 2003，2，1096-1103.

[6]Geho，D.H.，Lahar，N.，Ferrari，M.，Petricoin，E.F.，Liotta，L.A.，Biomed.Microdevices 2004，6，231-239.

[7]Desai，T.，Hansford，D.，Kulinsky，L.，Nashat，A.H.et al.，Biomed.Microdevices 1999，2，11-40.

[8]Desai，T.，Hansford，D.，Leoni，L.，Essenpreis，M.，Ferrari，M.，Biosensors and

Bioelectronics

2000，15，453-462.

[9]Hutchens，T.W.，Yip，T.T.，Rapid Commun.Mass.Spectrom.1993，7，576-580.

[10]Reddy G.，Dalmasso，E.A.，J.Biomed.Biotechnol.2003，4，237-241.

[11]Villanueva，J.，Philip，J.，Entenberg，D.，Chaparro，C.A.et al.，Anal.Chem.2004，76，1560-1570.

[12]Villanueva，J.，Philip，J.，Chaparro，C.A.，Li，Y.et al.，J.Proteome Res.2005，4(4)；1060-1072.

[13]Trauger，S.A.，Go，E.P.，Shen，Z.，Apon，J.V.et al.，Anal.Chem.2004，76，4484-4489.

[14]Go，E.P.，Apon，J.V.，Luo，G.，Saghatelian，A.et al.，Anal.Chem.，2005，77，1641-1646.

[15]Ferrari，M.，Nat.Rew.Cancer 2005，5，161-171.

[16]Pasqua，L.，Testa，F.，Aiello，R.，Nagy，J.B.，Madeo，G.，Phys.Chem.Chem.Phys 2003，5，640-645.

[17]Pasqua，L.，Testa，F.，Aiello，R.，Stud.Surf.Sci.Catal.2003，146，497.

[18]Hulmes，J.D.，Bethea，，D.，Ho，K.，Huang，S.P.et al，ClinicalProteomics Journal，2004，1，17-31.

[19]Diamandis E.P.，J.Natl.Cancer Inst.2004；96：353-6.

[20]Diamandis，E.P.，Mol.Cell.Proteomics 2004，3，367-78.

[21]Diamandis，E.P.，Clin.Cancer Res.2005，11，963-965.

[22]Koomen，J.M.，Shih，L.N.，Coombes，K.R.，Li，D.et al.，ClinCancer Res.2005，11，1110-1118.

[23]Ye，B.，Cramer，D.W.，Skates，S.J.，Gygi S.P.et al，Clin.CancerRes.2003，9，2904-2911.

Embodiment 2

Use nano-porous materials fractionated serum component

[0098] molecule that belongs to low-molecular-weight circulating protein matter group that many fronts do not characterize can provide the lasting physiopathologic lasting picture of biosome.Recently, comprise the protein group signal of low-molecular-weight molecule, identify by using mass spectrum to combine with the bioinformatics algorithm.These proofs have been pointed out to exist to comprise and have been enriched the news file that diagnostic message is originated.Attempting at present to sort and identify the molecule of supporting fingerprint.Such discovery---many this low-molecular-weight molecules may exist relevant with circulating carrier protein---may provide new chance for being in based on classification and isolation technics before the mass spectral analysis.

[0099] the nanoporous substrate has been represented and has been easy to and the classification of reproducible low-molecular-weight biological marker material and the new method of selective binding.The nano-structure porous silicon of aminopropyl-Bao quilt when being exposed to serum, can consume albumen serum, and produces the serum that different MS images changes.In addition, the aminopropyl-Bao with control aperture can be combined with the small set of haemocyanin by the nanoporous beaded glass, and discharges them under powerful wash-out.The protein of wash-out has different MS images, gel electrophoresis images, and different peptide sequences sign, and this size that is based on nano-pore changes.Two kinds of potential source biomolecules of oophoroma are learned mark, and transthyretin and aPoA-I are separated discriminatively based on the aperture of pearl.The system of this new serum stage division uses, and when combining with bioinformatic analysis, can increase the path of diagnostic message, and this uses countless histochemical stain agent with virologist in evaluation diseased tissue process is similar.The nano-porous materials surface can be used in those unsettled and carrier protein binding molecules in results and storing blood.In addition, the knowledge that obtains from these researchs relevant with the nanoporous substrate can promote to use the agent of infusibility nanometer results, and it can monitor the circulation about early stage disease association signal.

Brief introduction

[0100] with regard to its core, pathology are medical specialities, and it is based upon observes the careful systematicness of disease sign, by to the accurate comparison of normal and diseased tissue variant and discovery to some extent.Known to each medical student, the virgin state in pathological diagnosis field be when physical examination to the identification of four kinds of main states of cortical tissue's pathology (underlying tissue pathology): swollen (tumor), (dolor), heat (calor) and red (rubor) bitterly.Similarly, may observe beginning with vision and sense of touch to the initial inspection of tissue.Yet this just is used for the beginning of the modern times tissue assessment of complete pathological evaluation.Because the introducing of microscope and laboratory method has produced the practice of modern medicine pathology, technical progress has disclosed the sensitivity of the raising of lysis and deep observation.By modern clinical testing detection and organized processing, a large amount of tests provides instrument for contemporary virologist, so that significant diagnosis is provided for the doctor to its consulting.Be with all these tests, the form of distinguishing and explain sign and sign is the center of virologist's specialty.Some histology form, as given tissue to haematine and eosin (H﹠amp; E) different picked-up, with specific morbid state and prognosis result's correlativity be the process of sustainable development, it becomes the basis of pathology consulting.The histochemical method of tissue can be considered to the stage division of organizing of different dyes, antibody, probe etc.Yet the histochemical method of tissue has only disclosed the subcomponent of selected tissues, and allows other subcomponent to escape from detection.

[0101] continue that according to pathology new technology is integrated into it and put into practice this significant tradition, modern disease Neo-Confucianism is to estimate new test platform based on the applicability in patient's the diagnosis.On the one hand, mass spectrometer novel, that sensitivity is higher is used to excavate potential diagnostic signal in patient's body fluid and the tissue sample (Rosenblatt etc., 2004, see the list of references tabulation at embodiment 2 ends).These effort are parts of new subject, are called the tissue protein group, wherein generate the multiple combined measure of molecule in the patient tissue, and relevant with morbid state and result.Owing to use the method for composite marker that specificity and sensitivity are all effectively improved, himself show effect into swollen, bitterly, heat or red before, detect early and find that fatal disease is possible.

[0102] uses the mass spectral protein group fingerprint method that is associated with analysis of biological information, in the low-molecular-weight scope of blood and tissue protein group, produced about having clue (Chaurand and Caprioli, 2002 of potential diagnostic message content; Hingorani etc., 2003; Paweletz etc., 2000; Petricoin etc., 2002a; Petricoin etc., 2002b; Schwartz etc., 2004; Stoeckli etc., 2001; Yanagisawa etc., 2003).Since these news files are related to by these researchs, making great efforts is that basic components for the fingerprint of disease association checks order and characterizes.

[0103] uniting the molecule that forms the diagnosis image can be various small proteins and protein fragments, its flow in the interstitial fluid by a plurality of tissues and with blood vessel in cell set up balance.Continue synthetic and degrade proteins by cells in vivo, may react overall health status.The expressed protein group can be represented the mutual relationship of complexity between all cells (normal and disease cell) in the host, so body fluid, as serum, may be the abundant source of the exploitation information of the discovery of disease biomarker and diagnostic test method.Nearest work shows that the low-molecular-weight molecule that carries the disease fingerprint combines (Mehta etc., 2003) with bigger carrier molecule.So the separation of these low-molecular-weight molecules, and hold and protect them not to be subjected to the separation of the resident carrier protein that kidney removes is to be used for that the neoformation mark is found and the target based on mass spectral tissue protein group of order-checking.

[0104] may need to develop new stage division to analyzing of the low molecular weight protein (LMWP) that combines with carrier protein more comprehensively and widely, new method can disclose the potentially useful diagnostic message of using form now to obtain.The manufacturing on nano-porous materials surface can be a strategy of each subset of the albumen that occurs in the analysis cycle system systematically and protein fragments.Described nano-porous materials has the ability of being modified and being handled on the function by specific chemical derivatization process (derivitizataion), thereby the selectivity of making and self-defining combination and classification can be carried out.And nanoporous has significantly increased surface area, makes that selectively grading and combination can be so that greater efficiency and speed take place potentially.The method of selectively grading and material can have contribution to tissue protein group field, and this is similar to the innovation that clinical chemistry man is done in some sense in the process of the fixation of tissue of creating PAL use every day all over the world and histochemistry's reagent tool storage room (arsenal).

[0105] in the present embodiment, shown that the nano-porous materials surface is used as the instrument based on the analysis and the classification in the biomarker discovery of serum.In an experiment, the nanoporous Silicon Wafer is used for optionally exhausting the high-abundance proteins of serum.Another kind method is to collect different protein group section situations with beaded glass nanoporous, that the aperture is controlled from serum, to carry out follow-up wash-out and assessment.

Material and method

Blood serum sample

[0106] described blood serum sample is to be stored in-80 degrees centigrade, from normal donor's single merging thing.

Chemical reagent

[0107] chemical reagent: acetonitrile (acetonitrile (ACN)) (HPLC level), ammonium bicarbonate (NH ₄HCO ₃), (St.Louis MO) buys from Sigma-Aldrich company for iodoacetamide (97%), methyl alcohol (99+%) and dithiothreitol (DTT) (DTT).(Phillipsburg NJ) buys from Mallinckrodt Baker for formic acid (88%), acetic acid (ice).H ₂O is in the two distillations of indoor Kontes high-purity water system (Kontes High Purity Water System).The order-checking level is modified pig trypsase, and (Madison WI) buys from Promega.

[0108] other material: bovine serum albumin(BSA) is from Sigma-Aldrich company, St.Louis, and MO buys.

Ruby protein adhesive dyestuff is from Molecular Probes, Eugene, and OR buys.Prefabricated 4-12% Bis-tris 1D gel, LDS sample and race glue damping fluid, antioxidant and pre-staining benchmark albumen gradient are bought from Invitrogen company.Fused quartz (fusedsilica) is from Polymicro Technology Phoenix, and AZ buys.

The manufacturing of nanoporous Silicon Wafer

[0109] buy from Silicon Quest company, resistivity＜0.005ohm-cm's, boron doped, (100) directed Silicon Wafer is used as substrate.Wafer is at 120 ℃ piranha solution (H ₂SO ₄: H ₂O=1:1) cleaned 30 minutes in, subsequently at HF:H ₂Remove oxide in the hydrofluoric acid solution of O=1:10, and use deionized water rinsing.Porous silicon surface prepares in homemade Teflon element by electrochemical etching.Described wafer is placed on the bottom of Teflon element, and its underlay aluminium foil (0.1mm is thick) electrically contacts to provide.The platinum net is placed in the chamber of element as counter electrode.Electrolytic solution is 49% HF and the ethanol that volume ratio 1:1 mixes.Apply 72mA/cm ²Constant current density 65 seconds.After porous silicon forms,, and be placed in the vacuum to remove moisture immediately with this sample of deionized water rinsing.Described porous silicon is silylated in the toluene solution of 10%APTES (aminopropyltriethoxywerene werene).In order to remove bubble from nano-pore, described porous silicon was placed in the vacuum 3 minutes.Refluxed 3 hours in the reaction solution sealing pad at room temperature.Described porous silicon toluene, acetone rinsing several times, and at N ₂Dry in the air-flow.Barret-Joyner-Halenda (BJH) model that is applied to isotherm nitrogen desorb branch shows: pore diameter distribution is the center with 2-20nm.

Consume serum with the nano-structure porous silicon wafer segment

[0110] the nanoporous Silicon Wafer of aminopropyl bag quilt is placed in the 1.5ml test tube and rinse 4 times in deionized water.500 μ l combining anteserum samples with deionized water 1:5 dilution are added on the wafer subsequently, and with this potpourri temperature bath at room temperature 1.5 hours.After temperature was bathed, the supernatant of dilute serum was removed and is stored in-80 degrees centigrade, analyzed so that carry out the MS of back.For control group, the blood serum sample of dilution is frozen equally.As further control group, described wafer is also bathed with deionized water one equality of temperature that replaces dilute serum.

Collect albumen with the nanoporous beaded glass that the aperture is controlled

[0111] the controlled beaded glass in the aperture of aminopropyl bag quilt, the aperture is 70nm or 17nm, from Sigma company, St.Louis, MO buys.Nitrogen adsorption-desorb isothermal data show that the surface area of 17nm pearl is 30.8m ²/ g and pore volume are 0.032cm ³/ g.

[0112] for the 70nm pearl, surface area is 130.5m ²/ g and pore volume are 0.93cm ³/ g.

[0113], weighs the 10mg pearl and insert in the 1.5ml test tube for each experiment.Pearl deionized water rinse 4 times.Be applied to subsequently in the pearl sample with the combining anteserum of deionized water with 1:5 dilution, and this potpourri at room temperature temperature bathed 1.5 hours.After temperature was bathed, the supernatant of described dilute serum was removed and preserves.Twice of 1ml deionized water rinse of pearl.After the rinse, 500 μ l elution buffers (5ml acetonitrile, 5ml water, 10 μ l trifluoroacetic acids) are applied on the sample, and it at room temperature shook 0.5 hour subsequently.Afterwards, eluate is collected and is stored in-80 degrees centigrade, so that the MS that carries out subsequently analyzes.

Mass spectrum

[0114] spectrogram of low resolution mass spectrum generation is used as the agonic reading of vague generalization and removes to estimate grading effect.Surface-enhanced laser desorption ionization (SELDI) the chip Biomek 2000 biological processor (WCX2 of weak cation exchange

Ciphergen Biosystems Inc.) handles.The HCl that adds 100 μ l10mM is to chip, and then temperature was bathed 5 minutes.Then, HCl removes by suction and bathed 1 minute with 100 μ l water temperatures subsequently.Water is gone by suction and adds fresh water, carries out other 1 minute.Next add 100 μ l10mM ammonium acetates and 0.1%Triton X to the chip and temperature bathed 5 minutes.Described ammonium acetate potpourri is gone by suction subsequently and abandons, and next uses the warm once more bath of ammonium acetate potpourri 5 minutes.After these preparation process, described chip vacuum drying.5 μ l samples as serum, are added into chip point subsequently and go up also temperature bath 55 minutes.Described chip is used 150 μ l water rinses 3 times again with 150 μ l phosphate buffers earlier.This chip is subsequently by vacuum drying and add the solution of cinnamic acid in 50% (v/v) acetonitrile of 1.0 μ l30%, and 0.5% trifluoroacetic acid is added on each protein site twice, and dry between each the adding.This chip is used the PBS-II mass spectrum subsequently, and (Ciphergen Biosystems Inc.) analyzes.The spectrogram of each point in order under collection is set: detector voltage 1,800V; Focusing quality is 6,000Da; The quality upper limit (the hi mass limit) is 20,000Da; The susceptibility gain is made as 5; Laser intensity is 145; 15 parts of laser photos of each station acquisition; The numbering of position changes in 20 to 80 scope, and per 5 positions increase once.Create the flow process that to handle all samples with being equal to.

Protein sequencing research

[0115] protein analysis: the pearl classification sample that obtains from 17nm and 70nm hole pearl is by conventional Bradford assay determination, with bovine serum albumin(BSA) (BSA) standard of 200 μ g/mL in the 1mg/mL scope, under 595nm, the UV-VIS spectrophotometer (

Plus 384, Molecular Devices) upward monitor.

[0116] 1D gel separation and digestion: every kind of pearl classification sample 15g and the former serum of 3 μ L, dilution and boiled 5 minutes in 30 μ L LDS sample buffers at 95 ℃.This component goes up electrophoresis at 1D precast gel (4-12%Bis-tris), to separate the desired molecular weight zone of compound protein potpourri.Described gel ddH ₂O thoroughly cleans, and fixes 30 minutes in 50% methyl alcohol/10% acetum, uses Orchil dyeing is spent the night, and at burst of ultraviolel and before observing at ddH ₂Decolouring is 3 hours among the O.This gel is cut into 1mm ²Blob of viscose and described gel band in 50% methyl alcohol, decolour.The gel band reduces and alkylation with 10mM dithiothreitol (DTT) (DTT) and 55mM iodoacetamide, and temperature was bathed 1 hour in 4 ℃ trypsase (20ng/ μ L), and allows the 25mM NH at 37 ℃ ₄HCO ₃Middle digest spend the night (16 hours).In second day morning,, albumen is extracted from gel with the temperature bath repeatedly of 70% ACN/5% formic acid solution.

[0117] μ LC/MS/MS analyzes: described sample by freeze-drying near dry, and at the HPLC buffer A (95%H of 6.5 μ L ₂O, 5% ACN, 0.1% FA) the middle reconstruction, to carry out mass spectrophotometry.With (San Jose, CA) the LC Packings liquid chromatographic system of the Dionex of online connection carry out the anti-phase LC/MS/MS of microscopic capillary and analyze with the classical ion trap mass spectrometry of the ThermoFinnigan LCQ that has modified form nanometer injection source.Reverse phase separation is used inner, and the capillary column that is full of slurry carries out.C ₁₈The internal diameter of silica bound post is 75 μ m, and external diameter is 360 μ m, be the long fused quartz of 10cm that is filled with the 5 μ m pearls that have 300 dust holes (Vydac, Hesperia, CA).The μ of a 5mm-pre-column PepMap, C ₁₈The function of cylinder (cartridge (Dionex)) performance desalting column.Sample injects with μ L pickup mode, and washes 5 minutes with buffer A before linear gradient elution, and the linear gradient elution working concentration reaches 85% buffer B (95%ACN/5%H ₂O/0.1% formic acid), wash-out 95 minutes under the flow velocity of 200nL/min.Comprehensively after the MS scanning four MS/MS scanning (with data dependence pattern) to enriching most the peptide ion, and collision induced dissociation (collision induced dissociation (CID)) carries out under 38% collision energy, the ion injection electric is arranged on 2.00kV, and capillary voltage and temperature are respectively 22.80V and 180 ℃.

[0118] data analysis: data analysis is by Sequest Biowork Browser (ThermoFinnigan), in Swiss-Prot, the TrEMBL and Ensembl clauses and subclauses of the nonredundancy protein group of European bioinformatics research institute (European BioinformaticsInstitute), search MS/MS composes and carries out.After filtering out associated score (table 1) and the manual MS/MS of inspection data, peptide is considered to reasonable discovery (legitimate hits).The condition that is used for filtering data is the same strict with most of reference citations at least.

Table 1

Electric charge X _CorrDeltaCN ion Rsp

+1 >1.9 >0.1 >50％＝1

+2 >2.5 >0.1 >50％＝1

+3 >3.5 >0.1 >50％＝1

[0119] acceptable peptide finds need have X with respect to all other peptide in the database _CorrEvaluation=1.

The result

Consume serum with nano-structure porous silicon

[0120] classification serum strategy is the part of loss serum oneself protein, and the remaining albumen kind of subsequent analysis is seen Fig. 6.In order to attempt this method, produce a kind of nanoporous substrate from silicon.Having the nanoporous Silicon Wafer of asymmetric surface and the blood serum sample temperature of merging bathes.After 1.5 a hour temperature is bathed, described wafer be removed and serum in residual protein accept the MS assessment, see Fig. 7.The appearance of the new quasi-molecular ions pattern that is drawn by surperficial laser enhanced desorption ionization (surface enhanced laserdesorption ionization (SELDI)) mass spectrum results from classification technique.Whole spectrum is compared with independent not classification serum, demonstrates tangible difference.In the situation that consumes experiment, two peak significant changes in the MS spectrum.When with former blood serum sample, the blood serum sample that is classification is relatively the time, the peak that exhausts 8122m/z place in the sample significantly strengthens.On the other hand, when comparing, exhaust that the peak of 8927m/z significantly reduces in the sample with its main peak in the former blood serum sample.Therefore, temperature is bathed to follow and is removed the spectrum quality that the nanoporous particle has changed serum subsequently, and original small peak curve in the spectrum is enhanced to main intensity area.

Use the controlled glass in hole of nanoporous to carry out the molecule collection

[0121] as the replacement scheme of the protein group signal that measure to consume serum, develops the pearl collection strategy, be used for the molecular species of follow-up wash-out and analysis, see Fig. 8 with separation.The beaded glass of (17nm is to 70nm) aminopropyl bag quilt in different apertures and serum temperature are bathed, to carry out the selectivity molecular separation and to discharge.After bathing with the serum temperature, these pearl waters are gentle to be cleaned, and the molecule of combination subsequently is with harsh solution (harsh solution) wash-out.The kind of wash-out is analyzed by SEDLI MS subsequently, and compares with unassorted serum spectrum, sees Fig. 9.Compare with untreated serum, the macroscopic examination of 17nm pearl eluate has been disclosed unique MS spectrogram figure.And when the sample of relatively collecting and former (being untreated) serum, macroscopic examination is enough to detect the significant difference at the 7762m/z place.Although preponderate in the spectrogram of normal serum in the 8927m/z peak, the peak of 7762m/z is considered to the role of dominance in the subclass of collecting.

[0122] in order to estimate the effect of nano aperture in producing unique spectrum signal, the controlled beaded glass in the hole in 70nm aperture is used in the identical test.Surprisingly, use the controlled beaded glass in hole of larger aperture to produce visibly different SELDI MS spectrum, see Figure 10.At the 6629m/z place, a new main peak accounts for leading in the spectrum sample that wash-out from pearl goes out.Normal serum peak almost can not be differentiated at identical m/z place.On the contrary, 8927 peaks in the normal serum are considered to back burner the albumen kind of wash-out on than the macropore pearl.

[0123] will further characterize the character of the molecular components of wash-out on 17nm aperture pearl or the 70nm aperture pearl, 15 μ g albumen electrophoresis on 4-12%Bis-Tris SDS-PAGE gel in every kind of sample is then used

Figure 11 is seen in Ruby Red dyeing.In whole molecular weight spectrum, notice evident difference between the eluate of two types of pearls.The notable difference of noticing in this discovery and the analysis based on SELDI is consistent.Divide the analysis of subconstiuent that the further affirmation that pearl is differently divided subconstiuent in the classification serum is provided by the one dimension electrophoresis to eluate.

[0124] in order further to characterize the character between the eluate on 17nm aperture pearl and the 70nm aperture pearl, from the SDS-PAGE gel, downcut band, and with the protein component in these gel bands of trypsinization.The peptide section electrospray ionization mass spectrophotometry that produces.After data investigated to spectrum, peptide identity (peptide identity) was assigned on the composition of protein group of this gel, sees Figure 12.From the eluate of the pearl of 70nm pore size, identify 25 kinds of peptides, and from the eluate of the pearl of 17nm-pore size, identify 13 kinds of peptides.Although the peptide kind that identifies has some overlapping (six kinds of peptides have), observed difference, this shows tangible classification has taken place.Therefore, beaded glass with different apertures of three kinds of different the analysis showed that provides the method for separation of serum component.

Discuss

[0125] uses the Proteomic analysis (proteomic profiling) of mass spectrum together with the bioinformatic data method for digging, complicated and breathtaking news file have been disclosed, it is comprised in the low-molecular-weight zone of circulating protein matter group, and this may contain important diagnostic information.The nano-porous materials surface as the controlled beaded glass of Silicon Wafer and hole, provides in separation and the operation body fluid---such as the strategic new method of the low-molecular-weight biological information that is comprised in the serum.The system evaluation that nano-porous materials surface combination MS analyzes and biological information is interrogated, can disclose some fractionated scheme of the disease detection performance that has enhancing, discern the elements collection that is used for work-up that expands, and the easy and powerful method of the purifying of molecule own, separation and peptide/protein sequencing is provided.

[0126] last, use the nano-porous materials surface to carry out serum in this research and separate the remarkable change that can cause spectrogram, the 1-D electrophoretic analysis result of change and different sequence signs.In these parameters each all shows uses 17nm aperture pearl or 70nm aperture pearl to produce significant, unique classification.Although six kinds of peptides that ESI MS identifies are to use the pearl of arbitrary type to collect, the peptide of the uniqueness that only can separate with one type pearl is arranged.The peptide that is checked order comprises highly abundant plasma proteins and not too abundant kind.For example, in the pearl in 70nm aperture, isolated the albumen that is called as rna plymerase ii transcription regulatory factor the 8th subunit autoploid amboceptor.In the pearl component in 17nm aperture, histone 4 and Gorky's autoantigen have been collected.The appearance of the molecular species of the haemocyanin in these non-traditional understanding may have been supported following hypothesis: the fragment of biosome albumen enters serum, and potential describing of this biosome integral status is provided.With a kind of albumen that the pearl of 70nm pore size separates, apolipoprotein A-1 is reported as the potential sign (Zhang etc., 2004) of oophoroma recently.In addition, in the research of mentioning just now, transthyretin also is reported as the potential sign of oophoroma, is to collect with the pearl of 17nm pore size.

[0127] strengthen the use that from blood, obtains the special technique of biomarker information, may be similar with use particular tissues chemical research in the anatomy and pathology diagnosis.Anatomical pathology is semiotics (semiology), and its discriminating is to produce by the variance analysis between ill and normal structure sample.The sign of disease is by after a large amount of comparison directly perceived to the histotomy of normal dyeing, and by finding after the application of new organization chemical technology.The back is a kind of to be studied the method that pathology/organizational information excavate achievement is abundant sometimes, so that has promoted the recurrence of numerous disease and situation heavily to classify; Therefore, the diagnosis of many pathological conditions becomes and depends on mainly by the special research information revealed of histochemistry, sees Figure 13.Although surmounting the particular organization of conventional study analyzes---as H﹠amp; The E section---do not increase the message catalog kind (information content ofthe tissue) of tissue, but but it can increase acquired information significantly, cause the new improvement in classification of diseases and the prognosis prediction, and provide extra clue for disease origin and fundamental biological knowledge.

[0128] serum and circulating protein matter group can be the potpourris of high abundance and low abundance molecule, and these molecules have the most of biomarkers that carry important diagnostic information, and these information may be present in the low abundance zone of this concentration range.A challenge of clinical proteomics is; Develop the instrument that in the biological sample of complexity quick identification goes out these low abundance molecules.A kind of method is to develop liquid to flow by surface (flow through surfaces) or consumption structure (depletionarchitectures) quick to carry out, effective (robust) and easy classification and selectivity purifying.Nano-porous materials can carry out physical modification (physicomodification), and different whereby apertures and charge characteristic can be added to hole surface.These distinguishable features can allow the fine setting to albumen classification in the serum.In addition, rabphilin Rab can be affixed to material surface, with the classification performance of the described substrate of further improvement.

[0129] above-mentioned experiment shows and uses the material surface have nanoporous to carry out the spectrogram that the serum classification can produce obvious change, and this has shown the effect of stage division.A collecting device that purposes is a blood serum sample of described nano-porous materials.Along with the unstable molecule as biomarker in the serum becomes research interest place day by day, can begin standardized collection flow process.The nanoporous substrate can provide a kind of unstable micromolecular method of form chelating that is suitable for easily with the serum collection procedure.In such application, it is similar with special fixation of tissue agent that special nanoporous substrate becomes, and described fixing agent is selected according to selected situation, to preserve specific organizational attribution, so that carry out subsequently analysis and/or observation.

[0130] because silicon can be manufactured into the particle of micron-scale, the nanoporous substrate can be developed in the magnitude range of haemocyte.Such particle can be designed to have aperture and other physicochemical property of certain limit, to separate the low molecular weight carriers (low molecular weight cargo) that carry sufficient haemocyanin and they.As additional character, the particle of described micron-scale can be used identification tag coded, as metal label or quantum dot (quantumdots).This strategy can allow blood to be collected, and allows experience sorting or enrichment to handle separating particles from haemocyte, itself and Bruchez etc., 1998; Han etc., 2001; Nicewarner-Pena etc., the processing described in 2001 is similar.

[0131] originally studies show that: for low-molecular-weight molecule, based on the separation of nanoporous and purifying feasibility as a kind of methods of selectively grading, purification and analysis cycle protein group.

List of references

[0132]Bruchez，M.，Jr.，Moronne，M.，Gin，P.，Weiss，S.，andAlivisatos，A.P.(1998).Semiconductorna nocrystals as fluorescentbiological labels.Science 281，2013-2016.

Chaurand，P.，and Caprioli，R.M.(2002).Direct profiling and imagingof peptides and proteins from mammalian cells and tissue sections by massspectrometry.Electrophoresis 23，3125-3135.

[0133]Geho，D.H.，Lahar，N.，Ferrari，M.，Petricoin，E.F.，and Liotta，L.A.(2004).Opportunities for nanotechnology-based innovation in tissueproteomics.Biomed Microdevices 6，231-239.

[0134]Han，M.，Gao，X.，Su，J.，and Nie，S.(2001).Quantum-dot-tagged microbeads for multiplexed optical coding ofbiomoleculs.Nature Biotechnology 19，631-635.

[0135]Hingorani，S.R.，Petricoin，E.F.，Maitra，A.，Rajapakse，V.，King，C.，Jacobetz，M.A.，Ross，S.，Conrads，T.P.，Veenstra，T.D.，Hitt，B.A.，et al.(2003).Preinvasive and invasive ductal pancreatic cancer and itsearly detection in the mouse.Cancer Cell 4，437-450.

[0136]Howorka，S.，Cheley，S.，and Bayley，H.(2001).Sequence-specific detection of individual DNA strands using engineerednanopores.Nat Biotechnol 19，636-639.

[0137]Liotta，L.A.，Ferrari，M.，and Petricoin，E.(2003).Clinicalproteomics：written in blood.Nature 425，905.

[0138]Mehta，A.I.，Ross，S.，Lowenthal，M.，Fusaro，V.A.，Fishman，D.A.，Petricoin，E.，and Liotta，L.(2003).Biomarker amplification byserum carrier protein binding.Disease Markers 19，1-10.

[0139]Meller，A.，Nivon，L.，Brandin，E.，Golovchenko，J.，andBranton，D.(2000).Rapid nanopore discrimination between singlepolynucleotide molecules.Proc Natl Acad Sci USA 97，1079-1084.

[0140]Nicewarner-Pena，S.R.，Freeman，R.G.，Reiss，B.D.，He，L，Pena，D.J.，Walton，I.D.，Cromer，R.，Keating，C.D.，and Natan，M.J.(2001).Submicrometer metallic barcodes.Science 294，137-141.

[0141]Paweletz，C.P.，Gillespie，J.W.，Ornstein，D.K.，and al.，e.(2000).Rapid protein display profiling of cancer progression directly fromhuman tissue using a protein biochip.Drug Develop Res 49，34-42.

[0142]Petricoin，E.F.，3rd，Ornstein，D.K.，Paweletz，C.P.，Ardekani，A.，Hackett，P.S.，Hitt，B.A.，Velassco，A.，Trucco，C.，Wiegand，L.，Wood，K.，et al.(2002a).Serum proteomic patterns for detection of prostate cancer.J Natl Cancer Inst 94，1576-1578.

Petricoin，E.F.，Ardekani，A.M.，Hitt，B.A.，Levine，P.J.，Fusaro，V.A.，Steinberg，S.M.，Mills，G.B.，Simone，C.，Fishman，D.A.，Kohn，E.C.，and Liotta，L.A.(2002b).Use of proteomic patterns in serum to identifyovarian cancer.Lancet 359，572-577.

[0143]Rosenblatt，K.P.，Bryant-Greenwood，P.，Killian，J.K.，Mehta，A.，Geho，D.，Espina，V.，Petricoin，E.F.，3rd，and Liotta，L.A.(2004).Serum proteomics in cancer diagnosis and management.Annu Rev Med 55，97-112.

[0144]Schwartz，S.A.，Weil，R.J.，Johnson，M.D.，Toms，S.A.，andCaprioli，R.M.(2004).Protein profiling in brain tumors using massspectrometry：feasibility of a new technique for the analysis of proteinexpression.Clin Cancer Res 10，981-987.

[0145]Stoeckli，M.，Cháurand，P.，Hallahan，D.E.，and Caprioli，R.M.(2001).Imaging mass spectrometry：a new technology for the analysis ofprotein expression in mammalian tissues.Nat Med 7，493-496.

[0146]Wang，H.，and Branton，D.(2001).Nanopores with a spark forsingle-molecule detection.Nat Biotechnol 19，622-623.

[0147]Yanagisawa，K.，Shyr，Y.，Xu，B.J.，Massion，P.P.，Larsen，P.H.，White，B.C.，Roberts，J.R.，Edgerton，M.，Gonzalez，A.，Nadaf，S.，et al.(2003).Proteomic patterns of tumour subsets in non-small-cell lung cancer.Lancet 362，433-439.

[0148]Zhang，Z.，Bast，R.C.，Jr.，Yu，Y.，Li，J.，Sokoll，L.J.，Rai，A.J.，Rosenzweig，J.M.，Cameron，B.，Wang，Y.Y.，Meng，X.Y.，et al.(2004).Three biomarkers identified from serum proteomic analysis for thedetection of early stage ovarian cancer.Cancer Res 64，5882-5890.

Embodiment 3

[0149] the subscript formula is quoted in the present embodiment, referring to the list of references tabulation at embodiment 3 ends.

General introduction

[0150] the low-molecular-weight zone in the blood serum protein group (the low-molecularweight region of the serum/plasma proteome (LMWP)) just constantly obtains to pay close attention to as the potential source of medical diagnosis on disease label ^1-3(mass spectrometry (MS)) carries out serum LWM analysis of protein by mass spectrum ⁴Usually rely on surperficial laser enhanced desorb/ionization flight time (surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF)) ⁵, this comprises before to the mass spectrophotometry of the analyte of certain chip surface adsorption ^6-9But the high molecular that exists in the biological fluid, the interference of Abundant protein can limit the also repeatability of possibility impact analysis of susceptibility ¹⁰By before MS analyzes, using the equipment that allows specifically to catch the LMW polypeptide to improve the selectivity of analyzing based on MS, this interference can be reduced to minimum.In this piece of writing report, the nanoporous surface be used to optionally from human plasma, catch the LMW peptide (＜15,000Da).Use the auxiliary mastrix-assisted laser desorption ionization time of flight (matrix-assisted laser desor ption/ionization time-of-flight (MALDI-TOF)) of matrix as the method that detects and assess binding molecule, the peptide of collecting is carried out mass spectrophotometry (mass spectrometry (MS)).Because the selectivity that described analysis strengthens, realized to ng/mL concentration level in the human plasma little peptide (＜4, detection 000Da).

Result and discussion

[0151] purpose of this research work is to develop: based on size exclusion (size-exclusion) principle, separate the method for contained LMW peptide in the body fluid.Reach this target, the nanoporous surface with suitable porosity is used to carry out molecule and holds back.By using the thick silica nanometer perforated membrane bag of 500nm, make a kind of equipment by silicon.Use Lorentz-Lorenz model determination refractive index, determine the voidage of described film by ellipsometry (ellipsometry).The voidage of estimating is 57%.Use Brunauer-Emmett-Teller (cloth Long Naoer-Ai Meite-Teller BET) surface area of the film that temperature measurement such as nitrogen adsorption-desorb determines to be 670m ²/ g.Average pore size is about 7nm.Figure 14 has shown the form of silica nanometer perforated membrane under transmission electron microscope (Transmission Electron Microscopy (TEM)).

[0152] prepared chip is used to collect the LWM peptide from human plasma.After cleaning wetting described nanoporous surface in proper order with isopropyl alcohol and deionized water, (5 μ L) human plasma directly be added drop-wise to described chip surface and subsequently temperature bathe, to catch peptide/albumen kind.Carry out after a series of orders clean with deionized water, the acid MALDI matrix solution (alpha-cyano-4-hydroxycinnamic acid that contains high number percent organic solvent (50%v/v) by adding, α-cyano-4-hydroxycinnamic acid, CHCA) kind with combination discharges from described surface.The extract of a 1 μ L is placed on the MALDI dish and is used for MS and analyzes.Among Figure 15, between composing, the MALDI-TOF that the contrast surface that uses nanoporous silicon face and solid non-nano porous silica is obtained compares.In the former, detect about 70 kinds of low molecular weight peptides, comprise people's calcitonin, it has been mixed with the concentration of 1 μ g/mL when temperature is bathed in blood slurry.In contrast, from the MS spectrum that described control group surface analysis obtains, do not detect peptide.Use sinapic acid---a kind of matrix more special to large protein, the MALDI-TOF that identical nanoporous sample has been carried out replenishing analyzes.In this case, a spot of albumin and some other abundant plasma proteinss---they will account for leading in the MALDI-TOF spectrum in other mode in the extract although detected, but nearly all MS signal all concentrates on less than 15, in the zone of 000m/z.Therefore, 15kDa be nanoporosity by actual experiment condition and used surface obtain roughly hold back mass value.

[0153] although the present invention's bound by theory not, as shown in figure 15, the separation of LMW peptide and to detect may be because the specific pore of nanometer rate of described chip surface in the blood plasma.Have only those little, can be adsorbed onto described chip surface quantitatively to the analyte that can pass described hole easily.Therefore, this method can be used for enrichment and the analysis of LMW selectively.

[0154] described chip surface the absorption of analyte is needed enough strong, with in catching back opposing temperature bath step process to a large amount of flushings of chip.Under this concrete situation, LMW analyte and the ionic interaction that is present in the silanol groups on the described silica nanometer porous layer are the reasons of analyte combination.

[0155] repeatability of the spectrogram that produces in order to assess has been carried out replicate analysis five times to identical plasma sample.The MALDI-TOF spectrum that is obtained is presented among Figure 16, wherein can observe the repeatability of realizing the LMW spectrum.

[0156] detection limit of this method (detetion limit (DL)) can be by buying the peptide that obtains with the variable concentrations adding in human plasma, and calcitonin is estimated.People's calcitonin is doped before analysis and enters blood plasma, with the condition in the analogue body.This is analyzed by bathing the blood plasma through overdoping in described nano-stephanoporate silicon dioxide surface temperature, and MALDI-TOF subsequently analyzes and carries out.Figure 17 has shown corresponding to four kinds of variable concentrations levels, is low to moderate the peak intensity of the protonated molecule of calcitonin (m/z=3421.0 in theory) of 20ng/mL.In document ¹¹The data of the use similar approach of nearest report, this concentration detection limit has been represented significant improvement.

[0157] have a doping blood plasma of minimum calcitonin concentration---20ng/mL---, the absolute magnitude that contains calcitonin is 100pg (5 μ L adds to described nanoporous surface).Consider that 1/3 extraction solution is placed in analysis on the MALDI dish, the maximal value that MALDI-TOF detects is the 33pg calcitonin.An amount like this is corresponding to (standard peptide solution mixes with CHCA matrix, and 1 μ L places on the MALDI target and analyzes) under the standard conditions actual detected limit by the calcitonin of MALDI-TOF analysis.This shows: even under the least concentration of being analyzed, the calcitonin that major part is mixed is collected from blood plasma effectively by described nanoporous surface, and makes it can be used in the MS detection after the LMW peptide extracts.

[0158] the silicon-based nano porous surface also can produce such molecule and holds back.Nano-structure porous silicon is by the electrochemical etching manufacturing, and producing is situated between sees form (mesoscopic topography) (film of holes of nano size, similar with nano-stephanoporate silicon dioxide, cover minute sized silicon chamber).Described nano-structure porous silicon has been used as collection reagent in above-mentioned flow process, the chip of replacement of silicon dioxide bag quilt.Observe under the situation that strong excess large protein albumin exists the chelating of two kinds of low-molecular-weight standard peptide by mass spectrum.

Method

The manufacturing on nanoporous surface

[0159] described nanoporous oxidation film prepares as follows.8.71g surfactant EO ₁₀₆PO ₇₀EO ₁₀₆(pluronic (Pluronic) F127 BASF) is added in the 23g ethanol.Then, under high degree of agitation, add the 10g tetraethyl orthosilicate (tetraethylorthosilicate (TEOS), Aldrich), the potpourri of 0.1006g hydrochloric acid (20%), 10g ethanol and 10.629g water ¹⁸After stablizing 3-6 hour under the room temperature, precursor solution is rotated bag quilt (spin-coated) on described Silicon Wafer with 1900rpm, reaches 30s.After quilt was wrapped in rotation, described film toasted 12 hours down at 100 ℃, in stove, toasted 2 hours down in 400 ℃ subsequently.

Sample is prepared

[0160] described chip surface is wetting with isopropyl alcohol.After the washing, from healthy volunteer's 5 μ L human plasmas (according to disclosed criterion ¹⁹Collect) in its agreement and under the supervision of institutional review board (Institutional Review Board), be added to described chip surface to human experimenter's protection, and allow its temperature under room temperature, 100% humidity to bathe 30 minutes.Described sample shifts out with pipettor.Afterwards, should clean in proper order 5 times on the surface, allow water droplet on this surface, to stop 1 minute at every turn with every part 5 μ L water.After last the cleaning, to (v/v) contain 3mg/mL alpha-cyano-4-hydroxycinnamic acid (CHCA in the 1:1 potpourri at acetonitrile and 0.1% tetrafluoro acetate (TFA), Sigma) 3 μ L MALDI matrix solutions are used to extract the analyte that is attached to described chip surface.1 μ L extract is placed on the MALDI sample disc and allows its drying before mass spectrophotometry.

Mass spectroscopy

[0161] MALDI-TOF is at the Voyager-DE that is equipped with nitrogen laser luminous under 337nm ^TMCarry out on STR MALDI-TOF (Applied Biosystems) mass spectrometer.Used for 700 nanoseconds postponed extraction time and 20kV accelerating potential, under linear holotype, obtain spectrum.Usually average 500-600 opens laser image, to produce final sample spectrogram.

[0162] list of references

1.Liotta，L.A.，Ferrari，M.，Petricoin，E.Written in blood.Nature 425，905(2003).

2.Villanueva，J.，Tempst，P.OvaCheck：let′s not dismiss the concept.Nature 430，611(2004).

3.Villanueva，J.et al.Corretting common errors in identifyingcancer-specific serum peptide signatures.J.Proteome Res.4，1060-1062(2005).

4.Petricoin，E.F.et al.Use of proteomic patterns in serum to identifyovarian cancer.Lancet 359，572-577(2002).

5.Issaq，H.J.，Conrads，T.P.，Prieto，D.A.，Tirumalai，R.，Veenstra，T.D.SELDI-TOF MS for diagnostic proteomics.Anal.Chem.75，148A-155A(2003).

6.Ebert，M.P.，et al.Identification of gastric cancer patients by serumprotein profiling.J.Proteome Res.3，1261-1266(2004).

7.Zhang，Z.et al.Three biomarkers identified from serum proteomicanalysis for the detection of early stage ovarian cancer.Cancer Res.64，5882-5890(2004).

8.Chen，Y.D.，Zheng，S.，Yu，J.K.，Hu，X.Artificial neural networksanalysis of surface-enhanced laser desorption/ionization mass spectra ofserum protein pattern distinguishes colorectal cancer from h ealthypopulation.Clin.Cancer Res.10，8380-8385(2004).

9.Carrette，O.et al.Apanel of cerebrospinal fluid potentialbiomarkers for the diagnosis of Alzheimer′sdisease.Proteomics 3，1486-1494(2003).

10.Diamandis，E.P.Mass spectrometry as a diagnostic and acancer biomarker discovery tool：opportunities and potential limitations.Mol.Cell.Proteomics 3，367-378(2004).

11.Diamandis，E.P.，van der Merwe，D.E.Plasma protein profilingby masss pectrometry for cancer diagnosis：opportunities and limitations.Clin Cancer Res.11，963-965(2005).

12.Geho，D.H.，Lahar，N.，Ferrari，M.，Petricoin，E.F.，Liotta，L.A.Opportunities for nanotechnology-based innovation in tissue proteomics.Biomed.Microdevices 6，231-239(2004).

13.Trauger，S.A.et al.High sensitivity and analyte capture withdesorption/ionization mass spectrometry on silylated porous silicon.Anal.Chem.76，4484-4489(2004).

14.Go，E.P.et al.Desorption/ionization on silicon nanowires.Anal.Chem.77，1641-1646(2005).

15.Mehta，A.I.et al.Biomarker amplification by serum carrierprotein binding.Dis.Markers 19，1-10(2003-2004).

16.Liotta，L.A.et al.Importance of communication betweenproducers and consumers of publicly available experimental data.J.Natl.Cancer Inst.97，310-314(2005).

17.Lowenthal，M.S.，et al.，Clin.Chem.Oct 2005；51：1933-1945.

18.Cohen，M.H.，Melink，K.，Boiarski A.A.，Ferrari M.，Martin，F.J.Microfabrication of silicon-based nanoporous particulates for medicalapplications.Biomed.Microdevices 5，253-259(2003).

19.Hulmes，J.D.，et al.An Investigation of Plasma Collection，Stabilization，and Storage Procedures for Proteomic Analysis of ClinicalSamples.Clin.Proteomics 1，17-32(2004).

Embodiment 4

Collect and the new protedogenous protein group nano chips of target vascular therapy

[0163] quoting in the parenthesis in the present embodiment is referring to the list of references tabulation at embodiment 4 ends.

[0164] mass spectrum (mass spectrometry (MS)) is qualitative and quantitatively distinguishes the strong technology of molecular weight less than the peptide of 20kDa.Mention sensitivity, the laser desorption mass spectrum demonstrates the sensitivity (7) that can detect low vast mole (attamole) horizontal peptide recently.Therefore, in principle, MS allows to use only several microlitres can use sample, and the peptide that exists with the pg/mL level in solution is analyzed peek.Detect low-level peptide kind like this in human serum and the tissue, the extremely strong instrument of having represented biomarker to find.Yet the sensitivity of level can't be analyzed by the conventional MS to human serum and tissue and obtain like this.This mainly is because MS analyzes limited dynamic range, and it can under the condition as other albumen/peptide existence, detect the peptide analysis thing at maximum 10,000 times of excessive disturbance types under best situation.The interference of the high molecular carrier protein that exists with the mg/mL level allows to detect the peptides that exist than the maximum concentration that are low to moderate 3-4 the order of magnitude of magnitude, the i.e. detection of the peptide that exists in hanging down μ g/mL scope.

[0165] focus of current focus concentrate on low molecular weight protein group in the human serum (low-molecular weight proteomen (LMWP)) as the potential source of diagnosis marker (3,8-11).Use the LMWP that exists with low-down level in MS serum analysis and the tissue to mean: sample separation is necessary to the ability that strengthens us and detect this important information source widely.A kind of method that reduces the complexity of peptide to be analyzed is the part with special physiological chemical property of separating in the whole protein group, for example, based on size peptide is carried out fractionated.

[0166] has the absorption that the nano-stephanoporate silicon dioxide of certain pore size and voidage/silicon substrate surface allows low molecular weight peptide in the blood serum sample.Use has such surface generation molecule of deterministic nature and holds back or molecular sieve.Therefore a few microlitre serum can directly be added on the nanoporous surface, to collect LMWP.After cleaning described nanoporous surface, in conjunction with analyte can be collected, and by auxiliary laser desorption ionisation time-of-flight mass spectrometry (matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF the MS)) tracing analysis of matrix.By the surperficial sensitivity that improves specific big or small Protein Detection of use nanoporous that this method provides, the LMWP that exists with ng/mL concentration in allowing research serum and organizing.

[0167] catch and discern the sensitivity of the peptide that exists in serum and the tumor tissues as enhancing except developing the nanoporous particle, present embodiment is paid close attention to exploitation and is also improved integrated silica/silicon chip to improve sensitivity.The surface of described chip is built with some formation patterns " activity " nanoporous spot, and it is distributed on passivation, atresia, the non-adsorbable chip surface.The modification of described silica/silicon chip surface character, allow to be placed in the LMW peptide that contained in the sample of 5-10 μ L serum on the chip or tissue extract in the absorption of " spot " zone, concentrate and restriction.Before MS analyzed, the analyte that extracts combination in minimum volume (100-200nL) can strengthen sensitivity.And, from having the direct mass spectrum ionization of chip on described nanoporous point surface, can avoid further diluted sample.By in conjunction with concentration method on the chip (an on-chipup-concentration approach) and directly " spot " ionization, the raising in the sensitivity can surpass the analysis limit of existing pg/mL scope.

[0168] under standard conditions, (is applied on the object), crystal that unusual fraction produces only arranged by the actual bombardment of laser, and be used to obtain MS data (common 0.1-1%) as 1 μ L sample/matrix solution.Can be by adjusting the surface nature of described chip, obtain raising to the sensitivity of the purpose peptide analysis thing that concentrates on the surface, in advance.Silicon surface (silicon chip surfaces) can be fabricated, so that the atresia, passivation (the being non-adsorbable therefore) chip surface that present " activity " nanoporous spot of some formation patterns and be used for laser ionization.Such method allows to be placed in the LMW peptide that contains in the 5-10 μ L blood serum sample on the chip adsorbs in localized area (being nanoporous " spot ") and concentrates, and condition is that initial sample drop is that the nanoporous " spot " that surrounds with passivated surface is the center.Before MS analyzes, the analyte of combination being extracted with minimum volume (100-200nL), also is favourable to the sensitivity that strengthens detection of analytes.And, from the directly mass spectrum ionization of described nanoporous spot surface, can avoid further handling sample/dilute with matrix.The diameter of described nanoporous spot can be limited, perhaps by i) at binding peptide from described nanoporous surface extraction and be placed under the situation on the MALDI sample stage minimum volume that micropipettor can be operated; Ii) attempting under the situation of described chip direct ionization, the diameter of laser spots limits.In first kind of selection, can the nanoporous spot of test diameter in the 0.5-1mm scope, this requires to extract volume of solution in hundreds of is received the scope that rises.Under second kind of situation, can detect and be low to moderate the 0.1-0.2mm spot diameter.In conjunction with concentrating on the chip and directly " spot " ionization, can be for the detection of analytes of LMWP in serum or the tissue sample in analyzing provide the sensitivity of the order of magnitude (orders-of-magnitude) to improve, and the MS analysis limit that can promote these analytes is to below the ng/mL scope.

Test method

[0169] The manufacturing on nanoporous surface: described nanoporous oxidation film prepares as follows.8.71g surfactant EO ₁₀₆PO ₇₀EO ₁₀₆(pluronic (

) F127, BASF) be added in the 23g ethanol.Then, under high degree of agitation, add the 10g tetraethyl orthosilicate (tetraethylorthosilicate (TEOS), Aldrich), the potpourri of 0.1006g hydrochloric acid (20%), 10g ethanol and 10.629g water.After stablizing 3-6 hour under the room temperature, precursor solution is rotated coating (spin-coated) on described Silicon Wafer with 1900rpm, for the time 30s.After the rotary coating, described film 100 ℃ the baking 12 hours, subsequently in stove 400 ℃ the baking 2 hours.The thickness of film roughly is 500nm.The voidage of estimating be 57% and average pore size be about 7nm.Use definite Brunauer-Emmett-Teller (BET) surface areas of temperature measurement such as nitrogen adsorption-desorb, be estimated as 670m ²/ g.

[0170] The preparation of protein group chip: the process of silicon preparation comprises that 4 photolithography steps use the soft surface treatment that contacts with 1 time.After the standard clean step, the LASER SPECKLE zone is designed and with reactive ion etching method (Reactive Ion Etching, RIE, LamCl ₂: He=180:440sccm 100W 300mT) carry out etching.The thin titanium of 10nm/50nm/gold layer is restrainted volatilizer (Denton Vaccum, electric current 100mA, deposition 0.1nm/min) deposition with e-, and is formed pattern by rising of photoresist (photoresist) from process.Described nanoporous surface uses the described flow process of previous section to be rotated coating, and 400 ℃ of annealing.Described nanoporous surface is defined by photoetching process, and carries out etching with the buffer oxide etching agent.The point sample hole usefulness of described chip has negative charge, and photoresistance SU-8 (from MicroChemInc) makes.Expose, baking, expose back baking and flow process development with manufacturer's suggestion after, formed the SU-8 microstructure of 25 μ m.Oxygen plasma (MicroRIE 100W, O ₂100sscm) be used for polyglycol (PEG) and handle preceding cleaning.PEG is used to reduce the absorption of albumen on the nonporous silicon surface in concentration step in advance.This wafer is at the 1%PEG (M that contains triethylamine and silicon tetrachloride adjuvant _w1000Da is in toluene) middle adhesional wetting, to form covalent bond at silica surface.Poly-(dimethyl siloxane) (PDMS, Dow Corning) built elastomer-forming seal (elastometricstamp) in the mould with embossing pattern (pattern relief).This seal soaks in the toluene solution of 5% chlorine (dimethyl) octadecylsilane, drying, and contact with described substrate.50 ℃ of heated overnight also remove this seal.Linear octadecyl hydrocarbon chain (C ₁₈) provide good binding ability for the LASER SPECKLE zone.

[0171] Finishing: described chip is made up of independent hole, and each hole has two and has the zones of different that particular surface is formed.Described surface is at oxygen plasma (O ₂100sccm, 50W) in by hydroxylation.By the silanization 30 minutes at room temperature of the 0.5%v/v3-aminopropyltriethoxywerene werene (APTES) in isopropyl alcohol (IPA), with positive charge, amino this surface of introducing.Negative charge, 0.5%v/v3-sulfydryl propyl trimethoxy silicane (MPTMS) and the 0.5%v/v H of sulfenyl among the IPA ₂O is covered in this surface.Hydrophilic hydroxyl is handled with 1% polyglycol (PEG) that contains triethylamine and silicon tetrachloride adjuvant.Hydrophobic surface is by 5% chlorine (dimethyl) octadecylsilane (C ₁₈) (derivatized) of derivatization.Behind the silanization, this particle can be by cleaning 5 times in the solvent, and 110 ℃ of dryings 2 hours.(electric charge) and the functional group of physics can be measured.For example, the lip-deep quantity of electric charge that APTES handles can be measured by the measurement of zeta potential, and the amino density of functionalization can be passed through the Fmoc method, with for example 0.4ml piperidines, 0.4ml DCM and 1.6ml MeOH, measures.Untreated and hydrophobic nanoporous surface can be used as control group, studies the contribution of functional group to the LMWP enrichment.

[0172] The preparation of recombinant protein: because these experiments are the different evaluations first expressed in the mouse system of VEGF transcription, VEGF ₁₄₄And VEGF ₂₀₅ ^*Sequence cloned and confirmed.Prepare recombinant protein, analyze the VEGF of new evaluation ₁₄₄And VEGF ₂₀₅ ^*The functional activity of splicing variants.

[0173] The fractionated of low molecular weight protein (LMWP): described chip surface is wetting with isopropyl alcohol.After the washing, 5 μ L serum/tumor samples are added to described chip surface, and allow its temperature under room temperature, 100% humidity to bathe 30 minutes.Unnecessary sample removes with micropipettor.Afterwards,, deionization aseptic with every part 5 μ L, the water that does not contain the HPLC level of surfactant should clean 5 times on the surface in proper order, allow water droplet to stop 1 minute on this surface at every turn.After last the cleaning, to contain at (v/v) 3mg/mL alpha-cyano-4-hydroxycinnamic acid (CHCA in the 1:1 potpourri of acetonitrile and 0.1% tetrafluoro acetate (TFA), Sigma) 3 μ LMALDI matrix solutions are used to extract the analyte that is attached to described chip surface.1 μ L extract is placed on the MALDI sample disc and allows its drying before mass spectrophotometry.

[0174] Mass spectrum: MALDI-TOF is being equipped with the Voyager-DE that sends 337nm nitrogen laser ^TMCarry out on STR MALDI-TOF (Applied Biosystems) mass spectrometer.Use delay extraction time 700 nanoseconds and 20kV accelerating potential under linear holotype, to obtain spectrum.Usually 500-600 is opened laser image and average, to produce final sample spectrogram.

[0175] Protein sequencing research: protein analysis: the pearl classification sample that obtains from 17nm and 70nm hole pearl is by conventional Bradford assay determination, with 200 μ g/mL to interior bovine serum albumin(BSA) (BSA) standard of 1mg/mL scope, at 595nm, UV-VIS spectrophotometer

Plus 384, Molecular Devices) on monitor.

[0176] 1D gel separation and digestion: every kind of pearl classification sample 15g and the former serum of 3 μ L, dilution and boiled 5 minutes in 30 μ LLDS sample buffers at 95 ℃.This classification component goes up electrophoresis at 1D precast gel (4-12%Bis-tris), to separate the desired molecular weight zone of compound protein potpourri.Described gel ddH ₂O thoroughly cleans, and fixes 30 minutes in 50% methyl alcohol/10% acetum, uses

Orchil dyeing is spent the night, and at burst of ultraviolel and before observing at ddH ₂Decolouring is 3 hours among the O.This gel is cut into 1mm ²Blob of viscose and described gel band in 50% methyl alcohol, decolour.The gel band reduces and alkylation with 10mM dithiothreitol (DTT) and 55mM iodoacetamide, and temperature was bathed 1 hour in 4 ℃ trypsase (20ng/ μ L), and allows the 25mM NH at 37 ℃ ₄HCO ₃Middle digest spend the night (16 hours).In second day morning,, albumen is extracted from gel with the temperature bath repeatedly of 70%ACN/5% formic acid solution.

[0177] LC/MS/MS analyzes: described sample by freeze-drying near dry, and at the HPLC buffer A (95%H of 6.5 μ L ₂O, 5%ACN, 0.1%FA) the middle reconstruction is to be used for mass spectrophotometry.With (San Jose, CA) the LC Packings liquid chromatographic system of the Dionex of online connection carry out the anti-phase LC/MS/MS of microscopic capillary and analyze with the classical ion trap mass spectrometry of the ThermoFinnigan LCQ that has modified nanometer injection source.Reverse phase separation is used inner, and the capillary column that is full of slurry carries out.C ₁₈The internal diameter of silica bound post is 75 μ m, and external diameter is 360 μ m, be the long fused quartz of 10cm that is full of the 5 μ m pearls that have 300 dust holes (Vydac, Hesperia, CA).The μ of a 5mm-pre-column PepMap, C ₁₈Cylinder (cartridge) (Dionex) is brought into play the function of desalting column.Sample injects with μ L pickup mode, and washes 5 minutes with buffer A before linear gradient elution, and the linear gradient elution working concentration reaches 85% buffer B (95%ACN/5%H ₂O/0.1% formic acid), wash-out 95 minutes under 200nL/ minute flow velocity.Comprehensively after the MS scanning, be four MS/MS scanning (with data dependence pattern) to enriching most the peptide ion, and collision induced dissociation (collision induced dissociation (CID)) carries out under 38% collision energy, the ion injection electric is arranged on 2.00kV, and capillary voltage and temperature are respectively 22.80V and 180 ℃.

[0178] data analysis: data analysis is composed at Swiss-Prot, the TrEMBL of the nonredundancy protein group of European bioinformatics research institute (European BioinformaticsInstitute) and Ensembl clauses and subclauses search MS/MS by Sequest Biowork Browser (ThermoFinnigan) and is carried out.After filtering out associated score (seeing Table 2) and the manual MS/MS of inspection data, peptide is considered to rationally hit.The condition that is used for filtering data is the same strict with most of reference citations at least.

Table 2

Electric charge X _CorrDeltaCN ion Rsp

+1 >1.9 >0.1 >50％＝1

+2 >2.5 >0.1 >50％＝1

+3 >3.5 >0.1 >50％＝1

[0179] acceptable peptide hits, and need have X with respect to all other peptide in the database _CorrEvaluation=1.

[0180] list of references

1.Folkman，J.Fundamental concepts of the angiogenic process.CurrMol Med.2003 Nov；3(7)：643-51.

2.Affara NI，Robertson FM.Vascular endothelial growth factor as asurvival factor in tumor-associated angiogenesis.In Vivo.2004Sep-Oct；18(5)：525-42.

3.L.A.Liotta，M.Ferrari，E.Petricoin“Clinical Proteomics：Writtenin Blood”，Nature 425，301，October 30，2003.

4.Terracciano R.，Gaspari M.，Testa F.，Pasqua L.，Cuda G.，TagliaferriP.，Cheng M.C.，Petricoin E.F.，Liotta L.A.，Ferrari M.，Venuta S.，＂Selective Binding and Enrichment for Low Molecular Weight BiomarkerMolecules in Human Plasma after Exposure to Nanoporous SilicaParticles＂，Proteomics，Volume 6，Issue 11，Date：No.11 June 2006，Pages：3243-3250.

5.Gaspari M，Cheng MC，Terraccianol R，Liu X，Nijdam AJ，diFabrizio E，Petricoin EF，Liotta LA，Cuda G，Ferrari M，Venutal S，＂Nanoporous surfaces as harvesting agents for mass spectrometric analysisof peptides in human plasma＂J.Proteome Res.2006 May；5(5)：1261-6.

6.Ferrara N，Hillan KJ，Novotny W.B evacizumab(Avastin)，ahumanized anti-VEGF monoclonal antibody for cancer therapy.BiochemBiophys Res Commun.2005 Jul 29；333(2)：328-35.

7.Sunia A.Trauger，Eden P.Go，Z Shen，Junefredo V.Apon，Bruce J.Compton，Edouard S.P.Bouvier，M.G.Finn，and Gary Siuzdak，HighSensitivity and Analyte Capture with Desorption/Ionization MassSpectrometry on Silylated Porous Silicon＂Analytical Chemistry，76，4484-4489(2004).

8.E.F.Petricoin III et，al”Use of proteomic patterns in serum toidentify ovarian cancer”Lancet 359 572-77(2002).

9.D.Geho，N.Lahar，M.Ferrari，E.Petricoin，L.Liotta，“Opportunities for Nanotechnology-Based Innovation in TissueProteomics”，Biomedical Microdevices，Vol.6，No.3，September，2004，231.240.

10.M.Ferrari，Cancer Nanotechnology；Opportunities and Challenges，Nature Reviews，Cancer，Vol.5，n.3，pp.161-171，2005.

11.M.Ferrari，therapeutic microdevices and methods for making andusing sames，US patent 6,107,102.

12.Geho D，Cheng MC，Killian K，Lowenthal M，Ross S，Frogale K，Nijdam AJ，Lahar N，Herrmann P，Johann D，Whiteley G，Ferrari M，Petricoin E，LiottaL，＂Fractionation of Serum Components UsingNanoporous Substrates＂Bioconjug.Chem.2006 May-Jun；17(3)：654-61.

13.Walczak，et al.，in preparation.

14.Martin FJ，Melnik K，West T，Shapiro J，Cohen M，Boiarski AA，Ferrari M.Acute toxicity of intravenously administered microfabricatedsilicon dioxide drug delivery particles in mice：preliminary findings.DrugsR D.2005；6(2)：71-81.

15.Ferrari M.Nanovector Therapeutics，Current Opinions inChemical Biology，Vol.9，No.4，343-346，2005.

16.Cohen MH，Melnik K，Boiarski AA，Ferrari M，Martin FJ.Microfabrication of Silicon-Based Nanoporous Particulates for MedicalApplications，Biomedical Microdevices，Vol.5，No.3，September，2003.

17.M.Ferrari，therapeutic microdevices and methods for making andusing sames，US patent 6,107,102.

18.Decuzzi P，Lee S，Bhushan B，Ferrari M.A Theoretical Model forthe Margination of Nanoparticles within Blood Vessels.Annals ofBiomedical Engineering，Vol.33，No.2，February 2005，pp.179-190.

19.Nashat AH，Moronne M，Ferrari M.Detection of FunctionalGroups and Antibodies on Microfabricated Surfaces by ConfocalMicroscopy.Biotechnology and Bioengineering，Vol.60 No.2pp.137-146，1998.

20.M.Zhang，T.Desai，and M.Ferrari，“Proteins and Cells on PEGImmobilized Silicon Surfaces，”Biomaterials，Vol.19，1998，953-960.

21.J.K.Tu，T.Huen，R.Szema，and M.Ferrari，“Filtration ofSub-100nm Particles Using a Bulk-Micromachined，Direct-Bonded SiliconFilter”，Biomedical Microdevices，Vol.1，No.2，113-120，1999.

22.Nijdam AJ，Cheng MC，Geho DH，Fedele R，Herrmann P，KillianK，Espina V，Petricoin EP，Liotta LA，Ferrari M，＂PhysicochemicallyModified Silicon as Candidate Substrate for Protein Microarrays＂，Biomaterials.2007 Jan；28(3)：550-8.Epub 2006 Sep 20.

23.Crouch MF，Davy DA，Willard FS，Berven LA Insulin inducesepidermal growth factor(EGF)receptor clustering and potentiatesEGF-stimulated DNA synthesis in Swiss 3T3 cells：a mechanism forcostimulation in mitogenic synergy.Immunol Cell Biol.2000.78(4)：408-414.

24.Kute TE and Quadri Y.Measurement of proliferation nuclear andmembrane markers in tumor cells by flow cytometry.J.Histochem.Cytochem.1991.39(8)：1125-1130.

Embodiment 5

Be used to gather in the crops the nanoporous particle of proteolytic fragments

[0181] method that combines of development utilization nanometer technology and mass spectroscopy (MS) can be to protein group (the low-molecular weightproteome (LMWP) of the low molecular wt that exists in the serum of patients with mastocarcinoma;＜20kDa or＜know identification apace with new protein in 15kDa) entertain hope.The challenge of task can reduce the masking effect of bigger serum carrier protein like this, for example albuminous masking effect, and the more low-molecular-weight concentration of increase, lower abundance protein (lower abundance protein)---it is as the predictor (predictors) of individual patient for the prediction or the evaluation of the reaction of dissimilar treatments, for finding and for example diagnosing the illness that breast cancer is useful.In these researchs, developed chip based on the nanostructured silica/silicon, it has specific hole dimension and specific surface characteristics, for example or positive charge, or negative charge or hydrophobic character, it can strengthen the distinctiveness absorption of the protein in different molecular weight ranges, and these protein are present in from the serum that nude mouse separated that carries people's breast cancer knurl xenograft.Serum is separated from people MCF-7/ cyclooxygenase-2 (MCF-7/Cox-2) breast cancer knurl xenograft, compares the estradiol level that its generation doubly higher levels of prostaglandin E2 of 50-(PGE2) and 5-doubly increase with MCF-7/ vehicle Control cell.MCF-7/Cox-2 HBT cell is injected in the mammary fat pad female, all sizes of 8-10, ovariectomized nude mouse, the lasting release particles of the implanted beta estradiol of nude mouse.Behind tumor cell injection the 15th day, the 28th day, the 42nd day and the 60th day, blood is separated by cardiac puncture, and be processed into the separate blood sample, wash-out on nano chips, it is united with substance assistant laser desorpted ionized flight time Proteomic analysis and mass spectrum/microsequencing (MS/MS) subsequently.At identical time point, the tumor of breast xenograft is measured, and separatedly is used for Proteomic analysis and is used for Histological assessment.These studies have shown that the effectiveness that has the silica/silicon nano chips of particular surface feature in associating MS method, it can be to the low molecular weight protein in the serum of the mouse that carries the HBT allogeneic, provide repeatably the spectrogram with sensitivity to resemble, and be used for Sensitive Detection the protein that is produced in their early stage development, intrusion and transfer process by tumor of breast.So far, the LMWP of the serum that comes separating, discerned the protein of 79 kinds of uniquenesses from the mouse that has people's tumor xenogeneic graft.

Experimental design and method

[0182] according to below be used for tumor development and the program list set up, from mouse, collect test animal blood (TEST ANIMALS Blood).

[0183] sample comprises:

15 control mice---0.8-1.5ml blood (0.4-0.75ml serum)

15 carry 28 days mouse of HBT xenograft---0.8-1.5ml blood (0.4-0.74ml serum)

15 carry 42 days mouse of HBT xenograft---0.8-1.5ml blood (0.4-0.74ml serum)

15 carry 60 days mouse of HBT xenograft---0.8-1.5ml blood (0.4-0.74ml serum)

[0184] all analyses can be carried out at the serum of obtaining from blood sample.

Serum is collected and storage

[0185] blood can be obtained and be collected in the test tube of red lid by cardiac puncture, solidifies 2 hours in the time of 4 ℃, and removes grumeleuse.

[0186] after centrifugal, can be collected and branch such as branch such as quilt grade installs in the freeze pipe (cryoviles) from the serum of each mouse, in following being stored in-80 ℃.From the mouse of each time point, optionally be divided into three groups of A, B, C, every group comprises 5 mouse.After serum is collected, the A group (A1, A2, A3, A4, A5) in, the serum of the equal volume of collecting from every mouse is merged, and is stored with 100 μ l sample aliquot.In the animal of B group and C group, one group of similar sample can be collected.The serum that from group A, B, C, mixes, can be used to analyze and with the nano-beads incubation.All analyses can be carried out at the sample that directly thaws from freezing raw material, and the sample of the experience second time or repeated freezing thaw cycles can not be considered to the ingredient of these researchs.

Sample distributes and processing

[0187] the 400 μ l combining anteserums of Shou Jiing can be used to experiment.Remaining pooled serum is stored in-80 ℃, and is used for follow-up analysis.

The sample storage

[0188] after centrifugal, can be collected and be assigned in the freeze pipe from the serum of each mouse, in following being stored in-80 ℃ by whole branch.All analyses can be carried out at the sample that directly thaws from freezing raw material.The blood serum sample that appointment is used to process and analyze can transport on dry ice.

Carry out incubation (MW cutoff value 16kDa) with the nanoporous pearl

[0189] the former serum of sample aliquot (20 μ l) can be thawed, and diluted by the deionized water (1:5) (total sample volume 100 μ l) that adds 80 μ l.The serum of dilution can be at room temperature and 1 milligram nano-beads incubation one hour, and nano-beads is with deionized water prewashing 4 times, and (in the incubation process, with regard to pearl suspending liquid, sample can be monitored; And if needed, in the incubation process, every 15 minutes, nano-beads can be suspended in the serum of dilution again).After incubation, pearl can pass through desk-top microcentrifugation (30 seconds, 10,000rpm) separated from serum, the dilute serum that consumes through pearl can be removed, and is used for analyzing and/or-80 ℃ of storages; And pearl can be washed 3 times (each 5 minutes at normal temperatures) with the 0.1%TFA in deionized water of 100 μ l.In the end after the flushing, pearl can be at normal temperatures with acetonitrile/0.1%TFA (75:25) incubation of 80 μ l 30 minutes; And shift out eluate, the equal portions packing, and be stored under-80 ℃.For every kind of checked different nano-beads, can carry out this process.

[0190] each blood serum sample of Shou Jiing can carry out incubation with the different nano-beads prepared products that have 12 kinds altogether, and nano-beads comprises: the particle with 7nm or 20nm hole dimension; Has the different chemical oxide, NH ₂, the particle of PEG-NHS; And have the particle that diameter is 5 μ m or 20 μ m.The complete analysis of single blood serum sample with nano-beads of complete series can need the serum (12 pearls * 25 μ l serum/incubations) of about 300 μ l.

The SELDI of sample analyzes

[0191] for the blood serum sample of each merging, can obtain the SELDI-TOF mass spectrogram, and can compare with the serum (nanobead depleted serum) and the spectrogram of nano-beads eluate that nano-beads consumes.(WCX2 protein-chip, CiphergenBiosystems Inc), can be used to obtain the image (profiles) of conjugated protein to the weak cation exchange chip.Chip can be processed as described below.Under room temperature (room temperature (RT)), be placed on the chip in the bio-reactor, can be with the 10mM HCl incubation of 100 μ l 5 minutes.After HCl is removed in suction, at room temperature, with the deionized water rinsing chip spot secondary of 100 μ l, each one minute.Chip subsequently in the 10mM ammonium acetate of the 100 μ l that contain 0.1%Triton X-100 by the incubation secondary, each 5 minutes.Last ammonium acetate rinsing liquid is sucked away, and makes the chip drying.The sample of 5 μ l is applied on the chip spot subsequently; And at room temperature, in adding wet tank, incubation 55 minutes.Chip is done a rinsing with the deionized water of 150 μ l subsequently with 3 times (each 150 μ l) of phosphate buffered saline(PBS) flushing.After the drying, the cinnamic acid of 1.0 μ l30% in 50% (v/v) acetonitrile, 0.5% trifluoroacetic acid is employed secondary on each spot, carry out drying between using.After the drying, use PBS-II mass spectrometer (CiphergenBiosystems, Inc) checking chip.Obtained spectrogram under similarity condition is used to the purpose of comparison.Equipment setting below using can be collected spectrogram: use 1800 volts detector voltage, and 6000 daltonian focus quality (focus mass), the 20000 daltonian upper limits, sensitivity gain is set to 5, and laser intensity is 145.Under 5 positional increment, 15 laser photos (laser shot) (scope from 20 to 80) can be obtained in each position.

After former serum (with the dilution of the 1:5 deionized water) point sample of 5 μ l that will be similar dilution to the serum before the nano-beads incubation, can also collect the SELDI spectrogram, and from collecting the SELDI spectrogram, so that directly compare before the pearl incubation and the intensity and the contour shape of haemocyanin mass peak afterwards with 5 μ l dilute serums after every kind of nano-beads incubation (nano-beads dilution (consumption) serum).From the SELDI spectrum that every kind of nano-beads eluate (amounting to 4 μ l) of equal volume obtains, can compare with these spectrum.

The protein analysis of pearl eluate

[0192] the miniature protein test of use standard (Bradford or BCA test) adopts bovine serum albumin(BSA) (bovine serum albumin (BSA)) as the protein standard, can determine the protein concentration in the nano-beads.

The 1D gel separation

[0193] behind protein determination, sample can be analyzed by the 1D gel separation.Every kind of nano-beads eluate classification component of 15 μ g can be by incubation in the SDS sample buffer device of 30 μ l, and boils under 95 ℃ 5 minutes.Use precast gel (4-12% Bis-Tris), by the 1D electrophoresis, these classification components subsequently can be separated.After electrophoresis, gel can be rinsed in deionized water, fixes 30 minutes in 50% methyl alcohol/10% acetic acid solution, and uses Orchil dyes and spends the night.Before using Versadoc 3000 image analysis system imagings, gel can go to dye in deionized water 3 hours.The gel area that is lower than 20KDa can be detected, and use BioRad gel spot to cut the core that colloid system takes out select tape.The gel film that cuts out is placed in the compound plate of 96-hole.Gel film can be transferred on the CCC Protemics Core (bbk protein group sampling core analyzer) to be handled, and is used for the analysis undertaken by LC/MS/MS.

LC/MS/MS

[0194] gel film can be washed one hour in 50% methyl alcohol/5% acetic acid solution.Before gel film was dehydrated in acetonitrile, washing step can repeat once.Gel film can be rehydrated in the 10mM dithiothreitol (DTT) (DTT) in being in the ammonium bicarbonate of 0.1M, and at room temperature reduced 0.5 hour.Remove DTT solution, sample can be at room temperature, carry out alkylation with the 50mM iodoacetamide in the ammonium bicarbonate that is in 0.1M.Remove iodoacetamide reagent, gel pieces is in 5 minutes increments, in acetonitrile solution before the drying, and it can be with the flushing of 100mM ammonium bicarbonate.Gel pieces can be washed in 100mM ammonium bicarbonate 5 minutes before dewatering 5 minutes with acetonitrile once more.Gel can be dried 5 minutes.Before the 50mM ammonium bicarbonate that adds 20 μ l, by gel pieces was modified in the trypsase rehydrated 10 minutes in the order-checking level (sequencing grade) that is present in 25 μ l in the 50mM ammonium bicarbonate with 20 μ g/mL, force proteinase to enter in the gel pieces.Sample at 40 ℃ by incubation 6 hours.The peptide that forms washes secondary with the solution of acetonitrile/5% formic acid of 50%, and each 20 minutes, thus from polyacrylamide, be extracted out.These extracts are mixed in clean 96-hole compound plate, and are dried 90 minutes.

[0195] kapillary-liquid chromatography-Na spraying tandem mass spectrometry (Capillary-liquidchromatography-nanospray tandem mass spectrometry, Nano-LC/MS/MS), can on the TgermoFinnigan LTQ mass spectrometer that is equipped with the spraying source of receiving that operates in the positive ion mode, carry out.The LC system can be the UltiMate from LC-Packings A Dionex Co (sunnyvale, California) ^TMPlus system and Famous self-actuated sampler and Switchos post converter.Solvent orange 2 A can be that the water and the solvent B that contain 50mM acetate can be acetonitriles.Every kind of sample of 5 microlitres at first is injected into catches post (trappingcolumn) (LC-Packings A Dionex Co, Sunnyvale, California), and washes with 50mM acetate.Syringe port can be switched to injection, and peptide can be left away from the grabber wash-out, thereby arrives on the post.Be directly installed on receive the spraying top 5 centimetres, 75 millimeters ID ProteoPepII C18 post (New Objective, Inc.Woburn MA) can be used to chromatographic resolution.Peptide can be with the flow velocity of 300nl/min, uses the gradient of the B of 2-80%, directly, is washed 30 minutes and leaves away from post, thereby enter in the LTQ system.Amounting to working time is 58 minutes.Mass spectrometric scanning sequence can be designed to by sequencing: a complete scanning, the image magnified sweep of the electric charge of decision peptide, and the MS/MS scanning of enriching the place, peak of the maximum on spectrogram.Dynamically getting rid of (dynamic exclusion) can be used, and is used to get rid of the multiple MS/MS of identical peptide.

[0196] sequence information that comes from the MS/MS data can use the MascotDistiller that forms the peak tabulation and use the Turbo SEQUEST algorithm in BioWorks 3.1 softwares processed.Data handling procedure can be known according to Molec.Cell.Proteomics and carries out.The assignment peak has minimum 10 countings (S/N is 3).The exactness high in quality of precursor ion can be set as 1.5 dalton, to adapt to the attached selection of C13 ion; And fragment exactness high in quality can be set as 0.5 dalton.The modification of considering (variation) is methionine oxidation and urea groups methyl halfcystine.

Experimental result

I mass spectroscopy MALDI-TOF

What [0197] be used for these tests is the nanoporous pearl.

[0198] operable be 4 * 12 sample aliquot silica beads (4 kinds of different surface chemistry: silicon dioxide little with big hole, the hole that APTES and MPTMS are little).

The MALDI-TOF analytical plan

[0199] Cui Qu peptide mixes with alpha-cyano-4-hydroxy styrenes acid (α-cyano-4-hydroxycinnamic acid (CHCA)) solution, and this solution is the 4mg/mL solution in 50% (v/v) acetonitrile, 0.1%TFA.Independently test for each, the ratio of matrix/sample is specific.Sample/matrix solution of 1 μ l is put in stainless steel MALDI destination disk, and air drying.The time-delay extraction in, the MALDI-TOF spectrogram can

(applying biological system (AppliedBiosystems), Framingham obtain on MA) the STR mass spectrometer.Setting below linear positive ion mode uses: accelerating potential 20000V, grid voltage 91.5%, obtains mass range 800-20000m/z, laser intensity scope 2100-2300 at extraction 200 nanoseconds of time delay.400-1000 the mean value that single laser take a picture of each spectrogram in 100 chronophotograph series, obtaining.The peptide mixer of external unit adjustment use standard (the calimx 2+calimx of applying biological system 3) carries out every day.Linear 6-point calibration is used (about molecular weight (approx.Mw): 1300,2090,3600,5700,8100,11000 dalton).

The optimization that is used for the scheme of MS MALDI spot

[0200] three different samples/matrix ratio is tested: (i) 1:8; (ii) 1:4; And (iii) 5:3.Two kinds of standard peptide---feritin and calcitonin with 200ng/mL mix to the mouse control serum, and analyzed.It may be best test setting that three spectrograms in Figure 19 demonstrate the 5:3 ratio, considers following factor: (i) in the spectrogram that comes from mice serum can detected peak number; The (ii) overall absolute strength at peak; (iii) the mix signal to noise ratio (S/N ratio) (calcitonin and feritin) of peptide.

[0201] target is that the improved alternative scheme of sensitivity is also tested.Silicon dioxide aperture integument uses.After the flushing, the MALDI matrix solution of 4 μ l is added in the pearl, and the gained suspending liquid of 1.5 μ l is dripped on the MALDI Target Board by point the last time.With respect to the droplet prepared product of the drying of standard, resulting MALDI-TOF spectrogram produces outstanding signal intensity.

[0202] owing to the pearl that (i) is present in the matrix suspending liquid, may influence the homogeneity of matrix crystallization, and may make the exactness high in quality of measurement become poorer; (ii) in mass spectrometer, introduce silica dioxide granule, this may be because the deposition of these particles in the position of mass spectrometer inside (voltage grid etc.), make some performance losss of following the time, therefore determine the extraction conditions and the 5:3 sample/matrix ratio of employing standard, for use in the subsequent analysis of mice serum.

[0203] below this paper, reported the MALDI-TOF spectrogram of the control mice serum that obtains with described method.Carried out two kinds of different peptides and mixed,, thereby be used for comparing with other MALDI sample so that have additional reference peak, for example, with reference to Figure 20.Higher from the signal intensity ratio of calcitonin (100ng/mL is entrained in the mice serum) with the signal intensity that the preparation method of standard obtains.

With nanoporous pearl incubation control serum samples

[0204] takes following scheme, be used for the mice serum analysis.

[0205] pearl washes in advance: 4 * 100 μ l H ₂O.

[0206] incubation: at room temperature, with the dilute serum of 1 milligram pearl incubation 100 μ l, one hour.

[0207] flushing of pearl: 2 * 100 μ l H ₂O is subsequently in 0.1%TFA, with 100 μ l flushing once.

[0208] extraction: the 75/25CH of 80 μ l ₃CN/0.1%TFA, at room temperature incubation is 30 minutes.

[0209] preparation of MALDI: sample/matrix ratio is 5:3.

[0210] MALDI-TOF analyzes as describes in early days in this embodiment.

[0211] in Figure 21 shown spectrogram on the mouse control serum, use four kinds of different pearls to obtain, (silicon dioxide little with big hole, the hole that hole that APTES is little and MPTMS are little).

[0212] experiment of relevant repeatability

[0213] be the repeatability of assessment pearl prepared product, five parts of copy incubations of mouse control serum are carried out under use 5 * macroporous silica pearl kit condition.Copy for each incubation carries out twice MALDI-TOF analysis, produces 10 spectrograms.Automatically 28 maximum intensity peaks---is common for 10 all spectrograms---of being found by peak harvesting software (applying biological system (Applied Biosystems)) are used to statistical analysis.After relevant total signal strength (peak height) standardization, the variation factor of standardization intensity is calculated (28 peaks, 10 copys).The result reports in table 3, together also have obtained typical MS spectrogram.The average CV that relevant m/z measures is 0.02%.Average CV about peak height is 21.8.

[0214] peak height of ten replicated tests that table 3 silica beads LMWP collects and MS analyzes (5 repetition incubations, the MALDI-TOF of two repetitions analyzes).

Peak m/z	CV on m/zvalue	Peak heightIn exp 1	Peak heightI nex p2	Peak heightin exp 3	Peak heightIn exp 4	Peak heightIn exp 5	Peak heightIn exp 6	Peak heightIn exp 7	Peak heightIn exp 8	Peak heightIn exp 9	Peak heightIn exp 10	Averagepeak height	CV value
Peak m/z	CV on m/zvalue	Peak heightIn exp 1	Peak heightI nex p2	Peak heightin exp 3	Peak heightIn exp 4	Peak heightIn exp 5	Peak heightIn exp 6	Peak heightIn exp 7	Peak heightIn exp 8	Peak heightIn exp 9	Peak heightIn exp 10	Averagepeak height	CV value	905.4	0.05	491	541	528	470	398	393	475	502	462	476	473	10.2
1061.6	0.04	2623	2863	2768	2696	2844	2738	2655	3123	2168	2571	2705	9.1	905.4	0.05	491	541	528	470	398	393	475	502	462	476	473	10.2
1061.6	0.04	2623	2863	2768	2696	2844	2738	2655	3123	2168	2571	2705	9.1	1823.2	0.02	277	289	353	320	383	384	411	373	519	533	384	22.5
1979.5	0.02	2260	1897	2496	2625	3290	3293	2972	2740	2805	2655	2703	16.0	1823.2	0.02	277	289	353	320	383	384	411	373	519	533	384	22.5
1979.5	0.02	2260	1897	2496	2625	3290	3293	2972	2740	2805	2655	2703	16.0	2082.4	0.02	373	473	308	297	644	605	474	595	300	332	440	31.1
2482.5	0.02	51	44	72	61	74	79	85	67	65	69	67	18.6	2082.4	0.02	373	473	308	297	644	605	474	595	300	332	440	31.1
2482.5	0.02	51	44	72	61	74	79	85	67	65	69	67	18.6	2640.2	0.01	40	38	51	47	31	33	42	36	41	37	40	15.4
2707.9	0.02	62	51	59	68	52	44	59	48	46	40	53	17.0	2640.2	0.01	40	38	51	47	31	33	42	36	41	37	40	15.4
2707.9	0.02	62	51	59	68	52	44	59	48	46	40	53	17.0	27555	0.02	150	153	167	159	118	134	172	126	147	132	146	12.1
28225	0.01	31	29	45	38	37	40	42	36	46	40	38	14.4	27555	0.02	150	153	167	159	118	134	172	126	147	132	146	12.1
28225	0.01	31	29	45	38	37	40	42	36	46	40	38	14.4	3413.6	0.01	319	322	273	275	172	195	212	167	345	304	258	257
3497.9	0.01	137	141	118	121	74	86	83	76	142	116	109	24.9	3413.6	0.01	319	322	273	275	172	195	212	167	345	304	258	257
3497.9	0.01	137	141	118	121	74	86	83	76	142	116	109	24.9	3581.1	0.02	55	49	81	72	48	48	68	60	71	59	61	18.8
3907.3	0.02	41	39	46	42	70	69	55	46	84	76	57	28.9	3581.1	0.02	55	49	81	72	48	48	68	60	71	59	61	18.8
3907.3	0.02	41	39	46	42	70	69	55	46	84	76	57	28.9	4040.6	0.02	89	76	71	67	51	49	59	55	66	50	63	20.3
4061.8	0.02	688	615	511	641	410	418	544	446	460	402	513	20.3	4040.6	0.02	89	76	71	67	51	49	59	55	66	50	63	20.3
4061.8	0.02	688	615	511	641	410	418	544	446	460	402	513	20.3	4069.1	0.02	266	250	231	273	134	145	197	189	178	144	201	25.9
4104.8	0.02	175	160	127	133	107	120	72	106	120	104	122	24.0	4069.1	0.02	266	250	231	273	134	145	197	189	178	144	201	25.9
4104.8	0.02	175	160	127	133	107	120	72	106	120	104	122	24.0	4283.8	0.02	30	30	34	30	27	26	31	25	28	29	29	8.0
4360.9	0.02	24	30	40	29	23	26	36	28	41	37	31	21.2	4283.8	0.02	30	30	34	30	27	26	31	25	28	29	29	8.0
4360.9	0.02	24	30	40	29	23	26	36	28	41	37	31	21.2	4533.2	0.02	91	81	54	55	44	45	47	47	47	45	56	30.0
6785.5	0.03	36	38	54	38	26	28	31	28	66	56	40	34.7	4533.2	0.02	91	81	54	55	44	45	47	47	47	45	56	30.0
6785.5	0.03	36	38	54	38	26	28	31	28	66	56	40	34.7	6827.8	0.02	400	467	399	364	223	276	301	226	579	563	380	33.7
6996.2	0.02	148	174	165	134	79	87	96	80	218	199	138	37.0	6827.8	0.02	400	467	399	364	223	276	301	226	579	563	380	33.7
6996.2	0.02	148	174	165	134	79	87	96	80	218	199	138	37.0	81255	0.03	877	866	707	749	485	474	654	603	723	696	683	20.0
8212.2	0.02	180	195	154	127	102	108	67	110	156	158	136	29.1	81255	0.03	877	866	707	749	485	474	654	603	723	696	683	20.0
8212.2	0.02	180	195	154	127	102	108	67	110	156	158	136	29.1	8571.1	0.03	32	31	40	32	24	26	28	29	36	36	31	15.8
9069.6	0.03	54	60	48	39	30	31	32	33	40	41	41	25.4	8571.1	0.03	32	31	40	32	24	26	28	29	36	36	31	15.8

Peakm/z: peak m/z; The value of CV on m/v value:CV on m/v; The peak heights of Peak Heightinexp 1: embodiment 1; Peak Heightin exp2: the peak heights of embodiment 2; The peak heights of PeakHeightin exp 3: embodiment 3; Peak Heightin exp: the peak heights of embodiment 4; Peak Heightin exp 5: the peak heights of embodiment 5; Peak Heightin exp 6: the peak heights of embodiment 6; Peak Heightin exp 7: the peak heights of embodiment 7; The peak heights of Peak Heightinexp 8: embodiment 8; Peak Heightin exp 9: the peak heights of embodiment 9; Peak Heightin exp 10: the peak heights of embodiment 10; Average peak height: average peak height; CV value:CV value

II mass spectrum SELDI

[0215] The processing of serum of the animal of tumour is arranged from load: as described below, the sample of pooled serum is provided from 12 treated animals:

1. contrast pooled serum A

2. contrast pooled serum B

3. contrast pooled serum C

4. the 28th day trouble knurl serum (tumour of deriving from the clone 8 of MCF7 cell)

5. the 28th day trouble knurl serum (tumour of deriving from the clone 10 of MCF7 cell)

6. the 42nd day trouble knurl serum (tumour of deriving from the clone 8 of MCF7 cell)

7. the 42nd day trouble knurl serum (tumour of deriving from the clone 10 of MCF7 cell)

8. the 60th day seronegativity contrast (with the animal of matrigel (matrigel) injection)

9. the 60th day trouble knurl serum contrast 1 (tumour of deriving from clone BT-474)

10. the 60th day trouble knurl serum contrast 2 (tumour of deriving from baseline MCF7 cell line)

11. the 60th day trouble knurl serum (tumour of deriving from the clone 8 of MCF7 cell)

12. the 60th day trouble knurl serum (tumour of deriving from the clone 10 of MCF7 cell)

[0216] 6 group of pearl is used to carry out incubation with the blood serum sample that merges.Every group of pearl comprises 12 single pearl kits, and each of 12 combining anteserums is with a kit.The every group of pearl that uses is as follows:

Organize A.10 μ m diameter, aperture, silicon dioxide

Organize B.10 μ m diameter, aperture, the APTES derivatization, (+) electric charge

Organize C.10 μ m diameter, aperture, the APTES derivatization, (-) electric charge

Organize D.10 μ m diameter, macropore, silicon dioxide

Organize E.10 μ m diameter, macropore, the APTES derivatization, (+) electric charge

Organize F.10 μ m diameter, macropore, the APTES derivatization, (-) electric charge

[217] blood serum sample and following pearl kit incubation.Before with the serum incubation, all pearl kits at first are rinsed in hplc grade water 4 times.Blood serum sample is thawed and is diluted 1:5 with hplc grade water, and at room temperature, the dilute serum of 100 μ l and pearl kit incubation 1 hour.For example, the dilute serum sample 1 of 100 above-mentioned μ l (the control serum A of merging) carries out incubation with six groups of pearl type A to each group among the F, produces sample 1 to 6 (referring to table 4) respectively.Similarly, each 12 groups of blood serum sample that retain also with pearl group incubation independently, produce 72 samples (referring to table 4) altogether.

[0218] with the serum incubation after, integument precipitates, and shifts out the serum (depleted serum) through consuming.At room temperature subsequently, before the wash-out pearl, pearl is washed in hplc grade water 2 times in 0.1%TFA/ acetonitrile (25:75), flushing is 1 time and in 0.1%TFA.Final eluate (～80 μ l) is collected (having 72 samples altogether, referring to table 4) and is stored under-80 ℃, up to further processing.

The key word of table 4 pearl sample and eluate (sample number)

[0219] after incubation 5 days, eluate is thawed; And shift out the eluate of 60 μ l, and in SpeedVav, be concentrated to the cumulative volume of about 25 μ l, be used for the 1D gel analysis.The original eluate sample that concentrates eluate and retain all is stored under-80 ℃, up to accepting further processing, is respectively applied for 1D gel analysis and SELDI and analyzes.

[0220] all concentrated pearl eluates are mixed with the SDS-PAGE sample buffer, and separate on 8-16%Tris-glycocoll gradient gel and dye with Sypro ruby.After decolouring, use the BioRadVersadoc system to give gel imaging.In Figure 30, presented the gel result at Figure 22.

The eluate gel protein matter band excision thing that is used for tandem mass spectrum

[0221] the 1D SDS-PAGE gel of pearl eluate has shown that similar protein becomes band banding pattern (protein banding patterns), referring to Figure 22-27.With compare from the combining anteserum of three control-animal samples, the analysis of gel does not disclose any new protein belt, it belongs to from the serum of suffering from the knurl animal.Notice that also the protein heap(ed) capacity changes to some extent from the sample to the sample, the indivedual swimming lanes of some of them comprise obviously more the protein of a large amount (referring to gel 6, swimming lane 8 (sample 42 in the table 4).Based on these results, 90 bands are identified, and are used for cutting and submission, and adopt tandem mass spectrum to identify.

[0222] gel 6---and it separates eluate sample obtained from macropore MPTMS pearl, selectedly goes out, and is used for doing to analyze the most fully.Four low-molecular-weight bands are cut out from each swimming lane, with decision whether with protein form and change with pearl, and all main bands are present in (sample number 42 is corresponding to collected combining anteserum from the 42nd day clone 10 tumour load animals) in the swimming lane 8.The cutting of other low-molecular-weight band is from gel 4, and macropore APTES modifies pearl, swimming lane 12 and 13, and it corresponds respectively to from cloning 8 and clone 10 the 60th day collected combining anteserum of tumour load animal.At last, according to from the top of gel to the bottom, from the swimming lane 6 of gel 5, cut out additional band, it is corresponding to from the obtained eluate of aperture MPTMS pearl, sample number 27 (serum of collecting from the 28th day clone 10 animal).Band is identified, and uses BioRad Gel cutting robot (BioRad Gel cutting robot) cutting.The sample that cuts down is placed in the hole of microtiter plate in 96 holes, and is transferred into CCIC proteomics corer, is used for tandem mass spectrum.The pattern of gel cutting is presented among Figure 28,29 and 30, together with the index in the table 4.

[0223] the identification of proteins result who obtains from the tandem mass spectrum analysis of these samples is reported in other place of this embodiment by CCIC proteomics corer.

The SELDI of combining anteserum analyzes

[0224] the combining anteserum sample is moved out of from-80 ℃ of storages, melt, and the former serum of 150 μ l sample aliquot is moved out of, and the hplc grade water dilution by adding 600 μ l is to produce the blood serum sample of the dilution in 1: 5 that stores.The former serum of the dilution of 25 μ l is moved out of, snap frozen in liquid nitrogen, and be placed in-80 ℃ and store up to being used for the processing that SELDI analyzes.The WCX2 chip is filled in the bio-reactor, and at room temperature, the 10mMHCl with 100 μ l washed 5 minutes with sample point.Solution is sucked out, and spot washes 2 times with 100 μ lHPLC level water, each rinsing 1 minute.Spot washes 2 times with 10mM ammonium acetate+0.1%Triton X-100 of 100 μ l subsequently, each rinsing 5 minutes.After last damping fluid was sucked out, the WCX2 chip was shifted out from bio-reactor (Bioreactor), and air drying at room temperature.Dry WCX2 chip is placed in the bio-reactor and the dilute serum sample of 5 μ l is added in the sample point, and at room temperature, chip incubation 55 minutes in moist chamber.With blood serum sample incubation after 55 minutes, WCX2 chip spot is with the PBS flushing of 150 μ l 3 times.Each spot is used the hplc grade water rinsing of 150 μ l subsequently, and shifts out chip from bio-reactor, and at room temperature carries out air drying.Matrix (is in 1 μ l α among 50% acetonitrile and the 0.5%TFA-CHCA) and is applied in and is used for each spot, and be dried.Matrix is used and is repeated, and chip is analyzed in PBSII SELDI mass spectrometer.The SELDI spectrogram that obtains presents in Figure 31 to 33.

The SELDI of pearl eluate analyzes

[0225] from each pearl incubation thing, reclaims and next eluate material, use SELDIGoldChip to test.Eluate sample (1 μ l) is applied in the chip spot, and allows air drying.Matrix is added into as top description; And the device parameter below sample uses is analyzed.Eluate AU chip in the LMW scope is read parameter, with the same what be used for that WCX2 chip analysis scheme expressed.Sample sets such as the top identical sequence that is applied in the 1D SDS-PAGE gradient gel are analyzed.All 72 washings samples are with analyzed, and obtain two spectrograms.

The key word of the SELDI spectrogram of pearl eluate

[0226] sample number shows on the right side of spectrogram, is to refer to sample number, corresponding to those numbers that presented in table 4.Each pearl eluate is analyzed with two duplicate, as usefulness sample number X-1 and sample number X-2 institute clearly shows in the spectrogram.Be applied to the sample of each group of individual 1D gel, be applied to the sequence of gel and analyzed (referring to Figure 22 to 27).Therefore, Figure 34 A, 34B and 34c representative is applied to the SELDI spectrogram (Figure 22) of the eluate sample of gel 1; Figure 35 A, 35B and 35C representative are applied to the SELDI spectrogram (Figure 23) of the eluate sample of gel number 2; Figure 36 A, 36B and 36C representative are applied to the SELDI spectrogram (Figure 24) of the eluate sample of gel number 3; Figure 37 A, 37B and 37C representative are applied to the SELDI spectrogram (Figure 25) of the eluate sample of gel number 4; Figure 38 A, 38B and 38C representative are applied to the SELDI spectrogram (Figure 26) of the eluate sample of gel number 5; Figure 39 A, 39B and 39C representative are applied to the SELDI spectrogram (Figure 27) of the eluate sample of gel number 6.

III mass spectroscopy LC/MS (OSU CCIC)

In gel digestion and nanometer LC/MS/MS identification of proteins.

[227] 100 bands of total from 1D SDS-PAGE are digested, and analyze with nanometer LC/MS/MS.Detailed result is summed up in table 5.From checked 100 bands, identify 707 kinds of protein.Among them, 225 keratin (keratin) or keratin associated protein (keratin related proteins) are identified.Trypsase is used to digestion process, and therefore, trypsase (trypsin) and trypsase associated protein (trypsin relatedproteins) are also identified in sample, has 145 kinds of trypsase of total or trypsase associated protein to be identified out.At last, lysozyme is used as intrinsic standard (internal standard), to guarantee the performance of equipment; And identified 80 times (table 6).After removing, be 257 from the total number of the remarkable protein in 100 gel bands being identified corresponding to keratin, bacteriolysin and tryptic coupling thing.

[0228] as shown in table 5, most protein is repeatedly found in different swimming lanes/band.For example, haemoglobin β-1 chain has been identified several times in different swimming lane/bands.After only counting once, 154 many particular protein (referring to table 6) have been determined to every kind of protein ID (identity).Among them, 64 kinds are confirmed as keratin or keratin related protein, and 5 kinds are confirmed as trypsase or trypsase related protein, and 6 kinds are confirmed as lysozyme.Therefore, 79 kinds of unique serum proteins are confirmed (table 7) significantly.

[0229] scheme of recommending according to manufacturer, gel is digested with the order-checking level trypsase that comes from Promega (Madison WI), use Montage In-Gel Digestion Kit (digestion reagent box in the Montage glue) from Millipore (Bedford MA).In brief, band is put in order as far as possible near state, so that background polyacrylamide material minimizes; And be cut into 2mm * mm fragment.Gel pieces was washed one hour in 50% methyl alcohol/5% acetate subsequently.Gel pieces in acetonitrile by anhydrated close before, rinsing step is repeated once.Before adding was in 15mg/ml iodoacetamide in the 100mM ammonium bicarbonate soln, gel band was by hydration once more; And, use dithiothreitol (DTT) (dithiothertol (DTT)) solution (5mg/ml in 100mM ammonium bicarbonate) incubation 30 minutes.Before shifting out, make iodoacetamide and be with in the dark incubation 30 minutes.Gel band is used the circulation cleaning of acetonitrile and ammonium bicarbonate (100mM) again, carries out with 5 minutes increment spacings.After gel is dried in speed ratio Wei Ke (speed vac), by with they in the order-checking level modification trypsase of 50 μ l rehydrated 10 minutes, proteinase is sent in the gel pieces, and wherein order-checking level modification trypsase is present in the ammonium bicarbonate of 50mM with 20 μ g/mL.The ammonium bicarbonate of the 50mM of 20 μ l is added in the gel band subsequently, and potpourri at room temperature is incubated overnight.From the polyacrylamide gel fragment, acetonitrile with 50% and 5% formic acid extract peptide several times, and combine.The potpourri of extraction is concentrated to about 25 μ l in speed ratio Wei Ke, analyze to be used for nanometer LC/MS/MS.

[0230] kapillary-liquid chromatography-Na tandem mass spectrometry of spraying

(capillary-liquid chromatography-nanospray tandem mass spectrometry (Nano-LC/MS/MS)) can carry out on the TgermoFinnigan LTQ mass spectrometer in spraying source being equipped with to receive, and operates in the positive ion mode.The LC system can be the UltiMate with Famous self-actuated sampler and Switchos post converter of (LC-Packings ADionex Co, Sunnyvale, California) ^TMThe Plus system.Every kind of sample of 5 microlitres at first is injected into catches post (trapping column) (LC-Packings A Dionex Co, Sunnyvale, California), and washes with 50mM acetate.Then, syringe port can be switched to injection, and peptide can obtain wash-out from this capture and leave away, and enters into post.(New Objective, Inc.Woburn MA) can be used to chromatographic resolution to 5 centimetres, 75 microns ID ProteoPep II C18 post.Solvent orange 2 A is that the water and the solvent B that contain 50mM acetate are acetonitriles.Peptide is directly washed from this post and is left away with the flow velocity of 300nl/min, enters in the LTQ system.Gradient begins with 2% B, and B remained on 2% at initial 3 minutes.B was increased to 50% from 3-30 minute subsequently; And further be increased to 80% from 30-45 minute.B remains on 80% and continues 5 minutes, changes back to 2% then in 0.1 minute.Subsequently, before injection next time, post washed 14.9 minutes with 98% A.Amounting to working time is 65 minutes.Mass spectrometric scanning sequence is programmed, and is used for carrying out a full scan (full scan) and in the MS/MS scanning of spectrogram to 10 peptide peaks the abundantest.After detecting and carrying out MS/MS3 time of peptide, dynamically eliminating is used to get rid of the multiple MS/MS of same peptide.

[0231] processed from the sequence information of MS/MS data, be to use Mascot Batch with formation peak lists (peaklist) (.mgf file) with by using MASCOT MS/MS search (MASCOT MS/MS search) to carry out.Data processing; Carry out according to the guilding principle in Mole.Cell.Proteomics (molecular biology proteomics).The peak value of appointment has minimum 10 countings (S/N is 3).The exactness high in quality of precursor ion is set as 1.8Da, selects to adapt to the accidental of C13 ion; And fragment exactness high in quality is set as 0.5Da.The modification that is considered (variation) is methionine oxidation and urea groups methyl halfcystine.

Sample	Protein identity (ID)	Score value	Swiss-Prot registration number (Ascension)
Sample	Protein identity (ID)	Score value	Swiss-Prot registration number (Ascension)	A1	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	234	gi\|2781269

	Haemoglobin β	182	gi\|229301
	Haemoglobin β	182	gi\|229301		Haemoglobin β-1 chain [mouse (Mus musculus)]	176	gi\|1183932
	Alpha-globulin [homo sapiens (Homo musculus)]	74	gi\|28549		Haemoglobin β-1 chain [mouse (Mus musculus)]	176	gi\|1183932
	Alpha-globulin [homo sapiens (Homo musculus)]	74	gi\|28549	A2	Keratin 1[homo sapiens]	545	gi\|17318569
	Keratin 10[homo sapiens]	534	gi\|40354192	A2	Keratin 1[homo sapiens]	545	gi\|17318569
	Keratin 10[homo sapiens]	534	gi\|40354192		Haemoglobin β-1 chain [mouse]	499	gi\|1183932
	Haemoglobin β-2 chain [mouse]	297	gi\|1183933		Haemoglobin β-1 chain [mouse]	499	gi\|1183932
	Haemoglobin β-2 chain [mouse]	297	gi\|1183933		Alpha-globulin [mouse]	279	gi\|49900
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	235	gi\|2781269		Alpha-globulin [mouse]	279	gi\|49900
		235	gi\|2781269		Prediction: to keratin 6irs isomeride 12 similar [Canidae]	209	gi\|73996330
	Keratin 6B[homo sapiens]	162	gi\|21961227		Prediction: to keratin 6irs isomeride 12 similar [Canidae]	209	gi\|73996330
	Keratin 6B[homo sapiens]	162	gi\|21961227		Betaglobulin chain [homo sapiens]	141	gi\|66473265
	Platelet factor 4 [mouse]	87	gi\|13560695		Betaglobulin chain [homo sapiens]	141	gi\|66473265
	Platelet factor 4 [mouse]	87	gi\|13560695		Film is in conjunction with the sub-LytR[Bacillus cercus of transcriptional regulatory (Bacillus cereus) ATCC 10987]	56	gi\|42784428
A3	Haemoglobin β-1 chain [mouse]	509	gi\|1183932			56	gi\|42784428
A3	Haemoglobin β-1 chain [mouse]	509	gi\|1183932		Keratin 1[homo sapiens]	500	gi\|17318569
	Haemoglobin β-2 chain [mouse]	348	gi\|1183933		Keratin 1[homo sapiens]	500	gi\|17318569
	Haemoglobin β-2 chain [mouse]	348	gi\|1183933		Prediction: with keratin 1; Keratin-1; Cytokeratin-1; Hair alpha protein similar [chimpanzee (Pan troglodytes)]	265	gi\|55638031
	Keratin 10[rabbit (Oryctolagus cuniculus)]	258	gi\|87045985			265	gi\|55638031
	Keratin 10[rabbit (Oryctolagus cuniculus)]	258	gi\|87045985		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	197	gi\|2781269
	Prediction: to keratin 6irs isomeride 12 similar [Canidae]	172	gi\|73996330			197	gi\|2781269
	Prediction: to keratin 6irs isomeride 12 similar [Canidae]	172	gi\|73996330		Alpha-globulin [mouse]	171	gi\|49900
	The trypsase precursor	169	gi\|136429		Alpha-globulin [mouse]	171	gi\|49900
	The trypsase precursor	169	gi\|136429		Haemoglobin β subunit (HBB) (betaglobulin)	165	gi\|122643
	Betaglobulin chain [homo sapiens]	157	gi\|66473265		Haemoglobin β subunit (HBB) (betaglobulin)	165	gi\|122643
	Betaglobulin chain [homo sapiens]	157	gi\|66473265		Trypsinogen 10[mouse]	71	gi\|2358087
	Cytokeratin 9[homo sapiens]	66	gi\|435476		Trypsinogen 10[mouse]	71	gi\|2358087
	Cytokeratin 9[homo sapiens]	66	gi\|435476	A4	The trypsase precursor	211	gi\|136429
	Keratin 1, type II, cytoskeleton-mankind	112	gi\|7428712	A4	The trypsase precursor	211	gi\|136429
	Keratin 1, type II, cytoskeleton-mankind	112	gi\|7428712		Alpha-globulin [homo sapiens]	98	gi\|28549
	Lysozyme	96	gi\|229157		Alpha-globulin [homo sapiens]	98	gi\|28549
	Lysozyme	96	gi\|229157		Cytokeratin 9[homo sapiens]	72	gi\|435476
	Haemoglobin β subunit (HBB) (betaglobulin)	58	gi\|122606		Cytokeratin 9[homo sapiens]	72	gi\|435476
	Haemoglobin β subunit (HBB) (betaglobulin)	58	gi\|122606	A5	Haemoglobin β-1 chain [mouse]	515	gi\|1183932
	Haemoglobin β-2 chain [mouse]	351	gi\|1183933	A5	Haemoglobin β-1 chain [mouse]	515	gi\|1183932
	Haemoglobin β-2 chain [mouse]	351	gi\|1183933		The trypsase precursor	265	gi\|136429
	Alpha-globulin [homo sapiens]	147	gi\|28549		The trypsase precursor	265	gi\|136429

	Epidermal cell keratin 2[homo sapiens]	75	gi\|181402
	Epidermal cell keratin 2[homo sapiens]	75	gi\|181402		Lysozyme	65	gi\|229157
A6	Haemoglobin β-1 chain [mouse]	399	gi\|1183932		Lysozyme	65	gi\|229157
A6	Haemoglobin β-1 chain [mouse]	399	gi\|1183932		Keratin 1[homo sapiens]	370	gi\|7331218
	The trypsase precursor	263	gi\|136429		Keratin 1[homo sapiens]	370	gi\|7331218
	The trypsase precursor	263	gi\|136429		Alpha-globulin [mouse]	213	gi\|49900
	Keratin 10[homo sapiens]	192	gi\|40354192		Alpha-globulin [mouse]	213	gi\|49900
	Keratin 10[homo sapiens]	192	gi\|40354192		Lysozyme	67	gi\|229157
	Trypsinogen 10[mouse]	62	gi\|2358087		Lysozyme	67	gi\|229157
	Trypsinogen 10[mouse]	62	gi\|2358087	A7	Siderophillin [mouse]	304	gi\|17046471
	Keratin 1[homo sapiens]	244	gi\|17318569	A7	Siderophillin [mouse]	304	gi\|17046471
	Keratin 1[homo sapiens]	244	gi\|17318569		Interior alpha-trypsin inhibitor, heavy chain 4[mouse]	201	gi\|9055252
	The trypsase precursor	162	gi\|136429		Interior alpha-trypsin inhibitor, heavy chain 4[mouse]	201	gi\|9055252
	The trypsase precursor	162	gi\|136429		Gelsolin, endochylema-mouse	112	gi\|90508
	Rich histidine glucoprotein [mouse]	111	gi\|11066003		Gelsolin, endochylema-mouse	112	gi\|90508
	Rich histidine glucoprotein [mouse]	111	gi\|11066003		Thioredoxin [colon bacillus (Escherichia coli)]	98	gi\|148071
	Apolipoprotein A-I precursor-mouse	61	gi\|109571		Thioredoxin [colon bacillus (Escherichia coli)]	98	gi\|148071
	Apolipoprotein A-I precursor-mouse	61	gi\|109571		α-Jia Taidanbai	53	gi\|191765
A8	Cytokeratin 9[homo sapiens]	1282	gi\|435476		α-Jia Taidanbai	53	gi\|191765
A8	Cytokeratin 9[homo sapiens]	1282	gi\|435476		Keratin 1[homo sapiens]	1235	gi\|7331218
	Chain E, leech source property tryptase inhibitors trypsase compound	294	gi\|3318722		Keratin 1[homo sapiens]	1235	gi\|7331218
		294	gi\|3318722		Prediction: be similar to keratin 6irs isomeride 12[Canidae]	288	gi\|73996330
	Keratin 10[homo sapiens]	219	gi\|40354192			288	gi\|73996330
	Keratin 10[homo sapiens]	219	gi\|40354192		Epidermis keratin subunit II	130	gi\|293686
	Apolipoprotein A-I precursor-mouse	112	gi\|109571		Epidermis keratin subunit II	130	gi\|293686
	Apolipoprotein A-I precursor-mouse	112	gi\|109571		Lysozyme	97	gi\|229157
	Trypsinogen 7[mouse]	61	gi\|2358072		Lysozyme	97	gi\|229157
	Trypsinogen 7[mouse]	61	gi\|2358072		Short-chain dehydrogenase/reductase enzyme SDR[burkholderia (Burkholderia sp.) .383]	59	gi\|77965219
A9	Haemoglobin β-1 chain [mouse]	469	gi\|1183932			59	gi\|77965219
A9	Haemoglobin β-1 chain [mouse]	469	gi\|1183932		Haemoglobin β-2 chain [mouse]	448	gi\|1183933
	Keratin 1[homo sapiens]	293	gi\|17318569		Haemoglobin β-2 chain [mouse]	448	gi\|1183933
	Keratin 1[homo sapiens]	293	gi\|17318569		Alpha-globulin [mouse]	256	gi\|49900
	The trypsase precursor	208	gi\|136429		Alpha-globulin [mouse]	256	gi\|49900
	The trypsase precursor	208	gi\|136429		Prediction: to keratin 6irs isomeride 12 similar [dog class]	168	gi\|73996330
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	103	gi\|2781269			168	gi\|73996330
		103	gi\|2781269		Keratin 10[homo sapiens]	87	gi\|40354192
	Film is in conjunction with the sub-LytR[Bacillus cercus of transcriptional regulatory ATCC10987]	59	gi\|42784428		Keratin 10[homo sapiens]	87	gi\|40354192
		59	gi\|42784428	A10	Keratin 1[homo sapiens]	223	gi\|17318569
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	218	gi\|2781269	A10	Keratin 1[homo sapiens]	223	gi\|17318569
		218	gi\|2781269		The trypsase precursor	115	gi\|136429
	Lysozyme C (1,4-β-N-acetyl muramidase C)	94	gi\|47117006		The trypsase precursor	115	gi\|136429
	Lysozyme C (1,4-β-N-acetyl muramidase C)	94	gi\|47117006	A11	Type I keratin 16; K16[homo sapiens]	1301	gi\|1195531
	Keratin 14[homo sapiens]	1134	gi\|17512236	A11	Type I keratin 16; K16[homo sapiens]	1301	gi\|1195531
	Keratin 14[homo sapiens]	1134	gi\|17512236		Keratin type II	492	gi\|386849
	Keratin 6B[homo sapiens]	435	gi\|21961227		Keratin type II	492	gi\|386849

	Keratin 1[homo sapiens]	367	gi\|7331218
	Keratin 1[homo sapiens]	367	gi\|7331218		Keratin 5[homo sapiens]	349	gi\|4557890
	Prediction: to keratin 6irs isomeride 12 similar [dog class]	257	gi\|73996330		Keratin 5[homo sapiens]	349	gi\|4557890
		257	gi\|73996330		Prediction: similar to keratin 15 [ox]	209	gi\|61813798
	The trypsase precursor	179	gi\|136429		Prediction: similar to keratin 15 [ox]	209	gi\|61813798
	The trypsase precursor	179	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	149	gi\|2781269
	Prediction: to keratin 8 similar type II cytoskeleton-human bodies [chimpanzee]	90	gi\|55638407			149	gi\|2781269
		90	gi\|55638407		Cytokeratin 9[homo sapiens]	88	gi\|435476
	Trypsase 10[mouse]	66	gi\|2358087		Cytokeratin 9[homo sapiens]	88	gi\|435476
	Trypsase 10[mouse]	66	gi\|2358087	A12	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	204	gi\|2781269
	Insulin precurosor	178	gi\|136429	A12		204	gi\|2781269
	Insulin precurosor	178	gi\|136429		Epidermal cell keratin 2[homo sapiens]	72	gi\|181402
	Trypsinogen 10[mouse]	68	gi\|2358087		Epidermal cell keratin 2[homo sapiens]	72	gi\|181402
	Trypsinogen 10[mouse]	68	gi\|2358087	B1	The trypsase precursor	180	gi\|136429
	Apolipoprotein C-III mouse]	129	gi\|15421856	B1	The trypsase precursor	180	gi\|136429
	Apolipoprotein C-III mouse]	129	gi\|15421856		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	111	gi\|2781269
	The complement component C3 precursor	97	gi\|192392			111	gi\|2781269
	The complement component C3 precursor	97	gi\|192392		Trypsinogen 10[mouse]	54	gi\|2358087
B2	Keratin 1[homo sapiens]	195	gi\|17318569		Trypsinogen 10[mouse]	54	gi\|2358087
B2	Keratin 1[homo sapiens]	195	gi\|17318569		The trypsase precursor	176	gi\|136429
	Apolipoprotein C-III mouse]	124	gi\|15421856		The trypsase precursor	176	gi\|136429
	Apolipoprotein C-III mouse]	124	gi\|15421856		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	99	gi\|2781269
	The complement component C3 precursor	72	gi\|192392			99	gi\|2781269
	The complement component C3 precursor	72	gi\|192392		Trypsinogen 7[mouse]	67	gi\|2358072
	Trypsinogen 10[mouse]	62	gi\|2358087		Trypsinogen 7[mouse]	67	gi\|2358072
	Trypsinogen 10[mouse]	62	gi\|2358087	B3	The trypsase precursor	275	gi\|136429
	Apolipoprotein C-III[mouse]	129	gi\|15421856	B3	The trypsase precursor	275	gi\|136429
	Apolipoprotein C-III[mouse]	129	gi\|15421856		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	123	gi\|2781269
	The complement component C3 precursor	76	gi\|192392			123	gi\|2781269
	The complement component C3 precursor	76	gi\|192392		Trypsinogen 10[mouse]	68	gi\|2358087
B4	The trypsase precursor	265	gi\|136429		Trypsinogen 10[mouse]	68	gi\|2358087
B4	The trypsase precursor	265	gi\|136429		Apolipoprotein C-III[mouse]	125	gi\|15421856
	Lysozyme	74	gi\|229157		Apolipoprotein C-III[mouse]	125	gi\|15421856
	Lysozyme	74	gi\|229157		The complement component C3 precursor	71	gi\|192392
	Trypsinogen 10[mouse]	68	gi\|2358087		The complement component C3 precursor	71	gi\|192392
	Trypsinogen 10[mouse]	68	gi\|2358087	B5	The trypsase precursor	212	gi\|136429
	Apolipoprotein C-III[mouse]	120	gi\|15421856	B5	The trypsase precursor	212	gi\|136429
	Apolipoprotein C-III[mouse]	120	gi\|15421856		The complement component C3 precursor	75	gi\|192392
	Lysozyme	72	gi\|229157		The complement component C3 precursor	75	gi\|192392
	Lysozyme	72	gi\|229157		Trypsinogen 10[mouse]	62	gi\|2358087
B6	The trypsase precursor	176	gi\|136429		Trypsinogen 10[mouse]	62	gi\|2358087
B6	The trypsase precursor	176	gi\|136429		Keratin 1, type II, cytoskeleton-human body	142	gi\|7428712
	Apolipoprotein C-III[mouse]	122	gi\|15421856		Keratin 1, type II, cytoskeleton-human body	142	gi\|7428712
	Apolipoprotein C-III[mouse]	122	gi\|15421856		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	95	gi\|2781269
	The complement component C3 precursor	88	gi\|192392			95	gi\|2781269
	The complement component C3 precursor	88	gi\|192392		Trypsinogen 10[mouse]	66	gi\|2358087
B7	Albumin 1[mouse]	1536	gi\|29612571		Trypsinogen 10[mouse]	66	gi\|2358087
B7	Albumin 1[mouse]	1536	gi\|29612571		Keratin 1[homo sapiens]	1518	gi\|17318569
	Cytokeratin 9[homo sapiens]	649	gi\|435476		Keratin 1[homo sapiens]	1518	gi\|17318569

	The trypsase precursor	272	gi\|136429
	The trypsase precursor	272	gi\|136429		Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	259	gi\|73996330
	Complement component C3 precursor (HSE-MSF) [comprising: complement C3 β chain; Complement C3 α chain; C	99	gi\|1352102			259	gi\|73996330
		99	gi\|1352102		Trypsinogen 10[mouse]	66	gi\|2358087
	Putative protein matter SAV6338[Avid kyowamycin (Streptomycesavermitilis) MA-4680]	62	gi\|29832880		Trypsinogen 10[mouse]	66	gi\|2358087
		62	gi\|29832880	B8	Keratin 1[homo sapiens]	913	gi\|17318569
	Keratin 10[homo sapiens]	592	gi\|40354192	B8	Keratin 1[homo sapiens]	913	gi\|17318569
	Keratin 10[homo sapiens]	592	gi\|40354192		Cytokeratin 9[homo sapiens]	390	gi\|435476
	Haemoglobin β-1 chain [mouse]	350	gi\|1183932		Cytokeratin 9[homo sapiens]	390	gi\|435476
	Haemoglobin β-1 chain [mouse]	350	gi\|1183932		Haemoglobin, the β main chain [mouse] of being grown up	349	gi\|31982300
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	271	gi\|73996330		Haemoglobin, the β main chain [mouse] of being grown up	349	gi\|31982300
		271	gi\|73996330		The trypsase precursor	263	gi\|136429
	Prediction: similar to haemoglobin, type II cytoskeleton 5 (cytokeratin 5) is (CK 5) (58kDa cytokeratin (K5)	262	gi\|73996312		The trypsase precursor	263	gi\|136429
		262	gi\|73996312		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	260	gi\|2781269
	Type I keratin 16; K16[homo sapiens]	252	gi\|1195531			260	gi\|2781269
	Type I keratin 16; K16[homo sapiens]	252	gi\|1195531		Prediction: to keratin 1 isomeride 2 similar [ox]	243	gi\|76617876
	Haemoglobin β-2 chain [mouse]	217	gi\|1183933		Prediction: to keratin 1 isomeride 2 similar [ox]	243	gi\|76617876
	Haemoglobin β-2 chain [mouse]	217	gi\|1183933		Alpha-globulin [mouse]	152	gi\|49900
	Profilin 1[mouse]	111	gi\|56206029		Alpha-globulin [mouse]	152	gi\|49900
	Profilin 1[mouse]	111	gi\|56206029		Prediction: to trypsinogen 7 isomeride 4 similar [dog class]	102	gi\|73978531
	Guang PROTEIN C precursor [mouse]	98	gi\|11762010			102	gi\|73978531
	Guang PROTEIN C precursor [mouse]	98	gi\|11762010		Transthyretin [mouse]	95	gi\|56541070
	Type I keratin 10[lung fish]	85	gi\|57335414		Transthyretin [mouse]	95	gi\|56541070
	Type I keratin 10[lung fish]	85	gi\|57335414		Parvalbumin [mouse]	77	gi\|509139
	Lysozyme	73	gi\|841217		Parvalbumin [mouse]	77	gi\|509139
	Lysozyme	73	gi\|841217		Flp pili assembly protein CpaB family [Burkholderia belongs to E264]	57	gi\|83719123
	Haemoglobin β subunit (HBB) β (globulin)	55	gi\|122643			57	gi\|83719123
	Haemoglobin β subunit (HBB) β (globulin)	55	gi\|122643	B9	Keratin 10[homo sapiens]	1068	gi\|40354192
	Keratin 1[homo sapiens]	926	gi\|7331218	B9	Keratin 10[homo sapiens]	1068	gi\|40354192
	Keratin 1[homo sapiens]	926	gi\|7331218		Epidermal cell keratin 9[homo sapiens]	700	gi\|181402
	Cytokeratin 9[homo sapiens]	551	gi\|435476		Epidermal cell keratin 9[homo sapiens]	700	gi\|181402
	Cytokeratin 9[homo sapiens]	551	gi\|435476		Prediction: to keratin 6 IRSs similar [chimpanzee]	435	gi\|55638029
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	319	gi\|73996330		Prediction: to keratin 6 IRSs similar [chimpanzee]	435	gi\|55638029
		319	gi\|73996330		The trypsase precursor	237	gi\|136429
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	230	gi\|2781269		The trypsase precursor	237	gi\|136429
		230	gi\|2781269		Apolipoprotein C-III[mouse]	129	gi\|15421856
	The complement component C3 precursor	89	gi\|192392		Apolipoprotein C-III[mouse]	129	gi\|15421856
	The complement component C3 precursor	89	gi\|192392		Type I keratin 10[lung fish]	83	gi\|57335414
	Keratin 19[homo sapiens]	71	gi\|7594734		Type I keratin 10[lung fish]	83	gi\|57335414
	Keratin 19[homo sapiens]	71	gi\|7594734		Trypsinogen 10[mouse]	66	gi\|2358087
	Adenylate kinase [clonorchis sinensis]	59	gi\|22652628		Trypsinogen 10[mouse]	66	gi\|2358087
	Adenylate kinase [clonorchis sinensis]	59	gi\|22652628		Non-symbiosis haemoglobin [Alnus firma]	58	gi\|84993584
B10	Keratin 1[homo sapiens]	1448	gi\|17318569		Non-symbiosis haemoglobin [Alnus firma]	58	gi\|84993584
B10	Keratin 1[homo sapiens]	1448	gi\|17318569		Cytokeratin 9[homo sapiens]	1294	gi\|435476
	Keratin 6A[homo sapiens]	653	gi\|14250682		Cytokeratin 9[homo sapiens]	1294	gi\|435476
	Keratin 6A[homo sapiens]	653	gi\|14250682		Keratin 10[homo sapiens]	568	gi\|40354192

	Prediction: similar to keratin, type I cytoskeleton 14 (cytokeratin 14) is (CK14) isomeride 3[Bos (K14)	445	gi\|76649703
		445	gi\|76649703		Prediction: to keratin 6 IRSs similar [chimpanzee]	410	gi\|55638029
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	399	gi\|73996330		Prediction: to keratin 6 IRSs similar [chimpanzee]	410	gi\|55638029
		399	gi\|73996330		Type I keratin 16; K16[homo sapiens]	377	gi\|1195531
	Epidermis keratin subunit II	347	gi\|293686		Type I keratin 16; K16[homo sapiens]	377	gi\|1195531
	Epidermis keratin subunit II	347	gi\|293686		Keratin compound, acid, gene 14[mouse]	311	gi\|21489935
	Insulin precurosor	118	gi\|136429		Keratin compound, acid, gene 14[mouse]	311	gi\|21489935
	Insulin precurosor	118	gi\|136429		Prediction: to trypsinogen 7 isomeride 4 similar [dog class]	108	gi\|73978531
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	94	gi\|2781269			108	gi\|73978531
		94	gi\|2781269		The red pheasant (Gallus gallus) of type II alpha-Keratin IIB[]	89	gi\|46399075
	Prediction: similar to keratin, type II cytoskeleton 8 (cytokeratin 8) (A in the cytokeratin) [Rattus norvegicus]	87	gi\|62657929			89	gi\|46399075
		87	gi\|62657929		Trypsinogen 10[mouse]	66	gi\|2358087
	Short-chain dehydrogenase/reductase enzyme SDR[burkholderia 383]	66	gi\|77965219		Trypsinogen 10[mouse]	66	gi\|2358087
		66	gi\|77965219	B11	Complement C3 precursor (HSE-MSF) [comprises: complement C3 β chain; Complement C3 α chain; C	358	gi\|1352102
	Chain E, leech source property tryptase inhibitors trypsase compound	291	gi\|3318722	B11		358	gi\|1352102
		291	gi\|3318722		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	216	gi\|2781269
	Keratin 1 type II, cytoskeleton-people	175	gi\|7428712			216	gi\|2781269
	Keratin 1 type II, cytoskeleton-people	175	gi\|7428712		Keratin 15 [homo sapiens]	166	gi\|30583361
	Thioredoxin [Escherichia coli]	69	gi\|148071		Keratin 15 [homo sapiens]	166	gi\|30583361
	Thioredoxin [Escherichia coli]	69	gi\|148071		Trypsinogen 10[mouse]	66	gi\|2358087
B12	Keratin 10[homo sapiens]	872	gi\|40354192		Trypsinogen 10[mouse]	66	gi\|2358087
B12	Keratin 10[homo sapiens]	872	gi\|40354192		Keratin 1[homo sapiens]	827	gi\|17318569
	Keratin 6A[homo sapiens]	632	gi\|14250682		Keratin 1[homo sapiens]	827	gi\|17318569
	Keratin 6A[homo sapiens]	632	gi\|14250682		Keratin 17[homo sapiens]	516	gi\|48735384
	Cytokeratin 9[homo sapiens]	465	gi\|435476		Keratin 17[homo sapiens]	516	gi\|48735384
	Cytokeratin 9[homo sapiens]	465	gi\|435476		Prediction: similar to keratin, type I cytoskeleton 14 (cytokeratin 14) is (CK14) isomeride 3[Bos (K14)	444	gi\|76649703
	Keratin	437	gi\|386848			444	gi\|76649703
	Keratin	437	gi\|386848		Keratin K5	390	gi\|386850
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	310	gi\|73996330		Keratin K5	390	gi\|386850
		310	gi\|73996330		Keratin type 16	283	gi\|186685
	The trypsase precursor	275	gi\|136429		Keratin type 16	283	gi\|186685
	The trypsase precursor	275	gi\|136429		Calmodulin-analog 5[homo sapiens]	195	gi\|55859601
	Prediction: to keratin 17 similar [chimpanzee]	184	gi\|55644941		Calmodulin-analog 5[homo sapiens]	195	gi\|55859601
	Prediction: to keratin 17 similar [chimpanzee]	184	gi\|55644941		Type I keratin 10[lung angle]	84	gi\|57335414
	Prediction: similar to keratin, type II cytoskeleton 8 (cytokeratin 8) is (Keraton-8) (K8) [Homo] (CK8)	80	gi\|88988823		Type I keratin 10[lung angle]	84	gi\|57335414
		80	gi\|88988823		Glyceraldehyde-3-phosphate dehydrogenasa [homo sapiens]	62	gi\|31645
	Putative protein matter FG03380.1[maize Chi Chang (PH-1)]	57	gi\|46115076		Glyceraldehyde-3-phosphate dehydrogenasa [homo sapiens]	62	gi\|31645
	Putative protein matter FG03380.1[maize Chi Chang (PH-1)]	57	gi\|46115076	C1	Chain E, leech source property tryptase inhibitors trypsase compound	193	gi\|3318722
	Platelet basic protein matter [mouse]	156	gi\|13560694	C1		193	gi\|3318722
	Platelet basic protein matter [mouse]	156	gi\|13560694		Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	110	gi\|73996330
	Apolipoprotein A-II[mouse]	99	gi\|21618837			110	gi\|73996330
	Apolipoprotein A-II[mouse]	99	gi\|21618837		Ubiquitin	85	gi\|223061
C2	Keratin 10[rabbit]	288	gi\|87045985		Ubiquitin	85	gi\|223061
C2	Keratin 10[rabbit]	288	gi\|87045985		Keratin 1[homo sapiens]	224	gi\|17318569
	The trypsase precursor	179	gi\|136429		Keratin 1[homo sapiens]	224	gi\|17318569

	Blood platelet basis protein [mouse]	166	gi\|13560694
	Blood platelet basis protein [mouse]	166	gi\|13560694		Apolipoprotein A-II[mouse]	162	gi\|21618837
	Lysozyme	99	gi\|229157		Apolipoprotein A-II[mouse]	162	gi\|21618837
	Lysozyme	99	gi\|229157		Cytokeratin 9[homo sapiens]	75	gi\|435476
	Ubiquitin	65	gi\|223061		Cytokeratin 9[homo sapiens]	75	gi\|435476
	Ubiquitin	65	gi\|223061		Apolipoprotein C-I (Apo-CI) (ApoC-I)	63	gi\|114017
C3	The trypsase precursor	241	gi\|136429		Apolipoprotein C-I (Apo-CI) (ApoC-I)	63	gi\|114017
C3	The trypsase precursor	241	gi\|136429		Blood platelet basis protein [mouse]	160	gi\|13560694
	Apolipoprotein A-II[mouse]	105	gi\|21618837		Blood platelet basis protein [mouse]	160	gi\|13560694
	Apolipoprotein A-II[mouse]	105	gi\|21618837		Lysozyme	67	gi\|229157
	Ubiquitin	64	gi\|223061		Lysozyme	67	gi\|229157
	Ubiquitin	64	gi\|223061		Film is in conjunction with the sub-LytR[Bacillus cercus of transcriptional regulatory ATCC10987]	56	gi\|42784428
C4	The trypsase precursor	172	gi\|136429			56	gi\|42784428
C4	The trypsase precursor	172	gi\|136429		Blood platelet basis protein [mouse]	126	gi\|13560694
	Trypsinogen 10[mouse]	62	gi\|2358087		Blood platelet basis protein [mouse]	126	gi\|13560694
	Trypsinogen 10[mouse]	62	gi\|2358087		Apolipoprotein A-II[mouse]	61	gi\|21618837
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	57	gi\|2781269		Apolipoprotein A-II[mouse]	61	gi\|21618837
		57	gi\|2781269	C5	The trypsase precursor	167	gi\|136429
	Blood platelet basis protein [mouse]	127	gi\|13560694	C5	The trypsase precursor	167	gi\|136429
	Blood platelet basis protein [mouse]	127	gi\|13560694		Keratin 1[homo sapiens]	125	gi\|7331218
	Trypsinogen 10[mouse]	58	gi\|2358087		Keratin 1[homo sapiens]	125	gi\|7331218
	Trypsinogen 10[mouse]	58	gi\|2358087		Apolipoprotein A-II[mouse]	56	gi\|21618837
C6	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	251	gi\|2781269		Apolipoprotein A-II[mouse]	56	gi\|21618837
C6		251	gi\|2781269		The trypsase precursor	225	gi\|136429
	Platelet basic protein matter [mouse]	160	gi\|13560694		The trypsase precursor	225	gi\|136429
	Platelet basic protein matter [mouse]	160	gi\|13560694		Apolipoprotein A-II[mouse]	92	gi\|21618837
	Trypsase (EC3.4.21.4) precursor-Niu	88	gi\|67549		Apolipoprotein A-II[mouse]	92	gi\|21618837
	Trypsase (EC3.4.21.4) precursor-Niu	88	gi\|67549		Film is in conjunction with the sub-LytR[Bacillus cercus of transcriptional regulatory ATCC10987]	55	gi\|42784428
C7	Keratin 1[homo sapiens]	1151	gi\|17318569			55	gi\|42784428
C7	Keratin 1[homo sapiens]	1151	gi\|17318569		Cytokeratin 9[homo sapiens]	1009	gi\|435476
	Keratin 6C[homo sapiens]	539	gi\|17505189		Cytokeratin 9[homo sapiens]	1009	gi\|435476
	Keratin 6C[homo sapiens]	539	gi\|17505189		Keratin K5	375	gi\|386850
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	348	gi\|73996330		Keratin K5	375	gi\|386850
		348	gi\|73996330		Type II keratin Kb1[Rattus norvegicus]	319	gi\|57012354
	The trypsase precursor	224	gi\|136429		Type II keratin Kb1[Rattus norvegicus]	319	gi\|57012354
	The trypsase precursor	224	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	215	gi\|2781269
	Keratin 10[homo sapiens]	215	gi\|40354192			215	gi\|2781269
	Keratin 10[homo sapiens]	215	gi\|40354192		Interior alpha-trypsin inhibitor, heavy chain 4[mouse]	162	gi\|9055252
	α-Jia Taidanbai	61	gi\|191765		Interior alpha-trypsin inhibitor, heavy chain 4[mouse]	162	gi\|9055252
	α-Jia Taidanbai	61	gi\|191765		Thioredoxin [Escherichia coli]	61	gi\|148071
C8	Haemoglobin β-1 chain [mouse]	553	gi\|1183932		Thioredoxin [Escherichia coli]	61	gi\|148071
C8	Haemoglobin β-1 chain [mouse]	553	gi\|1183932		Haemoglobin β-2 chain [mouse]	493	gi\|1183933
	Haemoglobin, the β main chain [mouse] of being grown up	451	gi\|31982300		Haemoglobin β-2 chain [mouse]	493	gi\|1183933
	Haemoglobin, the β main chain [mouse] of being grown up	451	gi\|31982300		β-1 globulin [mouse]	432	gi\|4760586
	Unnamed protein [mouse]	352	gi\|12845853		β-1 globulin [mouse]	432	gi\|4760586
	Unnamed protein [mouse]	352	gi\|12845853		Alpha-globulin [mouse]	321	gi\|49900

	The trypsase precursor	245	gi\|136429
	The trypsase precursor	245	gi\|136429		Hemoglobin alpha subunit (hemoglobin alpha-chain) (alpha-globulin)	172	gi\|122474
	Beta Globulin chain [homo sapiens]	134	gi\|66473265			172	gi\|122474
	Beta Globulin chain [homo sapiens]	134	gi\|66473265		Beta Globulin [white collar actor monkey]	124	gi\|33415435
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	104	gi\|2781269		Beta Globulin [white collar actor monkey]	124	gi\|33415435
		104	gi\|2781269		Hemoglobin alpha subunit (hemoglobin alpha-chain) (alpha-globulin)	96	gi\|122405
C9	Apolipoprotein A-II[mouse]	228	gi\|21618837			96	gi\|122405
C9	Apolipoprotein A-II[mouse]	228	gi\|21618837		The trypsase precursor	186	gi\|136429
	Alpha-globulin [mouse]	138	gi\|49900		The trypsase precursor	186	gi\|136429
	Alpha-globulin [mouse]	138	gi\|49900		Blood platelet basis protein [mouse]	127	gi\|13560694
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	122	gi\|2781269		Blood platelet basis protein [mouse]	127	gi\|13560694
		122	gi\|2781269		Trypsinogen 10[mouse]	68	gi\|2358087
	Apolipoprotein C2[mouse]	67	gi\|817943		Trypsinogen 10[mouse]	68	gi\|2358087
	Apolipoprotein C2[mouse]	67	gi\|817943		Ubiquitin	64	gi\|223061
	Adenylate kinase [clonorchis sinensis]	55	gi\|22652628		Ubiquitin	64	gi\|223061
	Adenylate kinase [clonorchis sinensis]	55	gi\|22652628	C10	Keratin 1[homo sapiens]	1055	gi\|7331218
	Cytokeratin 9[homo sapiens]	508	gi\|435476	C10	Keratin 1[homo sapiens]	1055	gi\|7331218
	Cytokeratin 9[homo sapiens]	508	gi\|435476		Keratin 10[homo sapiens]	414	gi\|40354192
	Epidermal cell keratin 2[homo sapiens]	252	gi\|181402		Keratin 10[homo sapiens]	414	gi\|40354192
	Epidermal cell keratin 2[homo sapiens]	252	gi\|181402		Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	176	gi\|73996330
	The trypsase precursor	166	gi\|136429			176	gi\|73996330
	The trypsase precursor	166	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	129	gi\|2781269
	Epidermis keratin subunit II	86	gi\|293686			129	gi\|2781269
	Epidermis keratin subunit II	86	gi\|293686		Thioredoxin [Escherichia coli]	73	gi\|148071
	Trypsinogen 7[mouse]	69	gi\|2358072		Thioredoxin [Escherichia coli]	73	gi\|148071
	Trypsinogen 7[mouse]	69	gi\|2358072	C11	Apolipoprotein E	374	gi\|192005
	Keratin 1[homo sapiens]	303	gi\|7331218	C11	Apolipoprotein E	374	gi\|192005
	Keratin 1[homo sapiens]	303	gi\|7331218		The trypsase precursor	266	gi\|136429
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	167	gi\|73996330		The trypsase precursor	266	gi\|136429
		167	gi\|73996330		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	85	gi\|2781269
D1	The trypsase precursor	216	gi\|136429			85	gi\|2781269
D1	The trypsase precursor	216	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	104	gi\|2781269
	Lysozyme	83	gi\|229157			104	gi\|2781269
	Lysozyme	83	gi\|229157		Pancreas keratin 1[Rattus norvegicus]	65	gi\|6981420
	Putative protein matter LOC338797[homo sapiens]	55	gi\|70673359		Pancreas keratin 1[Rattus norvegicus]	65	gi\|6981420
	Putative protein matter LOC338797[homo sapiens]	55	gi\|70673359	D2	Chain E, leech source property tryptase inhibitors trypsase compound	139	gi\|3318722
	Epidermal cell keratin 2[homo sapiens]	93	gi\|181402	D2		139	gi\|3318722
	Epidermal cell keratin 2[homo sapiens]	93	gi\|181402		Trypsinogen 10[mouse]	64	gi\|2358087
D3	The trypsase precursor	279	gi\|136429		Trypsinogen 10[mouse]	64	gi\|2358087
D3	The trypsase precursor	279	gi\|136429	D4	Keratin 1[homo sapiens]	302	gi\|7331218
	Chain E, leech source property tryptase inhibitors trypsase compound	300	gi\|3318722	D4	Keratin 1[homo sapiens]	302	gi\|7331218
		300	gi\|3318722		Agnoprotein matter MGC:116262) [Rattus norvegicus]	190	gi\|71051822
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	179	gi\|73996330		Agnoprotein matter MGC:116262) [Rattus norvegicus]	190	gi\|71051822
		179	gi\|73996330		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	78	gi\|2781269
	Unnamed protein [japonica rice Japan is fine]	59	gi\|34906342			78	gi\|2781269
	Unnamed protein [japonica rice Japan is fine]	59	gi\|34906342	D5	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	256	gi\|2781269
	Keratin 1[homo sapiens]	230	gi\|7331218	D5		256	gi\|2781269
	Keratin 1[homo sapiens]	230	gi\|7331218		The trypsase precursor	178	gi\|136429
	Cytokeratin 9[homo sapiens]	121	gi\|435476		The trypsase precursor	178	gi\|136429
	Cytokeratin 9[homo sapiens]	121	gi\|435476		Trypsinogen 10[mouse]	68	gi\|2358087

	Trypsinogen 7[mouse]	68	gi\|2358072
	Trypsinogen 7[mouse]	68	gi\|2358072		Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC10987]	56	gi\|42784428
D6	The trypsase precursor	211	gi\|136429			56	gi\|42784428
D6	The trypsase precursor	211	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	178	gi\|2781269
	Trypsinogen 7[mouse]	70	gi\|2358072			178	gi\|2781269
	Trypsinogen 7[mouse]	70	gi\|2358072		Trypsinogen 10[mouse]	66	gi\|2358087
D7	Keratin 6A[homo sapiens]	491	gi\|14250682		Trypsinogen 10[mouse]	66	gi\|2358087
D7	Keratin 6A[homo sapiens]	491	gi\|14250682		Keratin 6B[homo sapiens]	435	gi\|21961227
	Prediction: similar to keratin, type I cytoskeleton 14 (cytokeratin 14) is (CK 14) isomeride 3[Bos (K14)	372	gi\|76649703		Keratin 6B[homo sapiens]	435	gi\|21961227
		372	gi\|76649703		Keratin K5	359	gi\|386850
	Cytokeratin 9[homo sapiens]	317	gi\|435476		Keratin K5	359	gi\|386850
	Cytokeratin 9[homo sapiens]	317	gi\|435476		Keratin 1[homo sapiens]	312	gi\|7331218
	The trypsase precursor	259	gi\|136429		Keratin 1[homo sapiens]	312	gi\|7331218
	The trypsase precursor	259	gi\|136429		Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	251	gi\|73996330
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	144	gi\|2781269			251	gi\|73996330
		144	gi\|2781269		Thioredoxin [Escherichia coli]	79	gi\|148071
	Countertrypin=myosin type trypsin inhibitor [mouse, blood plasma, partial peptide, 20aa, in 4 fragments the 4th]	73	gi\|407619		Thioredoxin [Escherichia coli]	79	gi\|148071
		73	gi\|407619		Countertrypin=myosin type trypsin inhibitor [mouse, blood plasma, partial peptide, 23aa, in 4 fragments the 1st]	64	gi\|407613
	Trypsinogen 10[mouse]	62	gi\|2358087			64	gi\|407613
	Trypsinogen 10[mouse]	62	gi\|2358087		Myosin [mouse]	60	gi\|2546995
D8	Haemoglobin, the β main chain [mouse] of being grown up	324	gi\|31982300		Myosin [mouse]	60	gi\|2546995
D8	Haemoglobin, the β main chain [mouse] of being grown up	324	gi\|31982300		The trypsase precursor	260	gi\|136429
	Alpha-globulin [mouse]	140	gi\|49900		The trypsase precursor	260	gi\|136429
	Alpha-globulin [mouse]	140	gi\|49900		Apolipoprotein C-III[mouse]	129	gi\|15421856
	The complement component C3 precursor	102	gi\|192392		Apolipoprotein C-III[mouse]	129	gi\|15421856
	The complement component C3 precursor	102	gi\|192392		Lysozyme	66	gi\|229157
	Apolipoprotein A-II[mouse]	64	gi\|21618837		Lysozyme	66	gi\|229157
	Apolipoprotein A-II[mouse]	64	gi\|21618837		Trypsinogen 10[mouse]	64	gi\|2358087
	Non-symbiosis haemoglobin [Alnusfirma]	64	gi\|84993584		Trypsinogen 10[mouse]	64	gi\|2358087
	Non-symbiosis haemoglobin [Alnusfirma]	64	gi\|84993584	D9	The trypsase precursor	178	gi\|136429
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	116	gi\|2781269	D9	The trypsase precursor	178	gi\|136429
		116	gi\|2781269		Trypsinogen 10[mouse]	69	gi\|2358087
	Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC10987]	55	gi\|42784428		Trypsinogen 10[mouse]	69	gi\|2358087
		55	gi\|42784428	D10	Keratin 1[homo sapiens]	500	gi\|17318569
	Interior alpha-Keratin inhibitor, heavy chain 4[mouse]	400	gi\|9055252	D10	Keratin 1[homo sapiens]	500	gi\|17318569
	Interior alpha-Keratin inhibitor, heavy chain 4[mouse]	400	gi\|9055252		The trypsase precursor	261	gi\|136429
	Prediction: to keratin 4 isomeride 2 similar [ox]	222	gi\|76617900		The trypsase precursor	261	gi\|136429
	Prediction: to keratin 4 isomeride 2 similar [ox]	222	gi\|76617900		Type II keratin Kb18[Rattus norvegicus]	182	gi\|57012352
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	175	gi\|73996330		Type II keratin Kb18[Rattus norvegicus]	182	gi\|57012352
		175	gi\|73996330		Keratin compound 2, alkalescence, gene 1[mouse]	165	gi\|6678643
	Type II keratin Kb1[Rattus norvegicus]	165	gi\|57012354		Keratin compound 2, alkalescence, gene 1[mouse]	165	gi\|6678643
	Type II keratin Kb1[Rattus norvegicus]	165	gi\|57012354		Prediction: similar to keratin 25A [dog class]	163	gi\|73965965
	Cytokeratin 9[homo sapiens]	150	gi\|435476		Prediction: similar to keratin 25A [dog class]	163	gi\|73965965
	Cytokeratin 9[homo sapiens]	150	gi\|435476		Larva keratin XLK[Africa xenopus]	141	gi\|13111394
	Thioredoxin [Escherichia coli]	141	gi\|148071		Larva keratin XLK[Africa xenopus]	141	gi\|13111394

	Keratin, type II cytoskeleton 4 (cytokeratin-4) be (keratin-4) (K4) (cytoskeleton 57kDa keratin) (CK-4)	135	gi\|82654948
		135	gi\|82654948	Three-D space structure-the S2 of escherichia coli thioredoxin, 2.8 dust resolution	133	gi\|230335
	TPA:TPA_exp: keratin Kb40[mouse]	125	gi\|46485130		133	gi\|230335
	TPA:TPA_exp: keratin Kb40[mouse]	125	gi\|46485130	Interior alpha inhibitor H4 heavy chain [Rattus norvegicus]	116	gi\|9506819
	Prediction: to keratin 24 isomeride 1 similar [ox]	112	gi\|76644680	Interior alpha inhibitor H4 heavy chain [Rattus norvegicus]	116	gi\|9506819
	Prediction: to keratin 24 isomeride 1 similar [ox]	112	gi\|76644680	Unnamed protein [mouse]	109	gi\|26324736
	TPA:TPA_exp: type II keratin Kb36[mouse]	103	gi\|46485128	Unnamed protein [mouse]	109	gi\|26324736
	TPA:TPA_exp: type II keratin Kb36[mouse]	103	gi\|46485128	Type I keratin 15 [lung fish]	102	gi\|57335394
	Thioredoxin 1 (TRX1) is [luminous bacillus subsp.laumondii TTO1] (TRX)	100	gi\|36787919	Type I keratin 15 [lung fish]	102	gi\|57335394
		100	gi\|36787919	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	100	gi\|2781269
	LOC495267 protein [Africa xenopus]	98	gi\|54261576		100	gi\|2781269
	LOC495267 protein [Africa xenopus]	98	gi\|54261576	Putative protein matter LOC77055[mouse]	98	gi\|85701680
	Keratin 6 IRS 3[homo sapiens]	96	gi\|27901522	Putative protein matter LOC77055[mouse]	98	gi\|85701680
	Keratin 6 IRS 3[homo sapiens]	96	gi\|27901522	Thioredoxin [Fei Shi vibrios ES114]	90	gi\|59478765
	Zgc:92061[zebra fish]	90	gi\|49902693	Thioredoxin [Fei Shi vibrios ES114]	90	gi\|59478765
	Zgc:92061[zebra fish]	90	gi\|49902693	Keratin 9[dog class]	79	gi\|62122767
	Phosphoserine phosphatase [having a liking for bitter ancient bacterium DSM9790]	79	gi\|48431085	Keratin 9[dog class]	79	gi\|62122767
		79	gi\|48431085	Pancreas proteinase 1[mouse]	78	gi\|6981420
	Zgc:92035[zebra fish]	74	gi\|49904349	Pancreas proteinase 1[mouse]	78	gi\|6981420
	Zgc:92035[zebra fish]	74	gi\|49904349	Prediction: similar to keratin, type I cytoskeleton 18 (cytokeratin 18) is (CK18) [ox] (K18)	71	gi\|76617986
	Keratin 19[pheasant]	70	gi\|45384356		71	gi\|76617986
	Keratin 19[pheasant]	70	gi\|45384356	Keratin compound 2, alkalescence, gene 8[mouse]	70	gi\|13624315
	Trypsinogen 10[mouse]	62	gi\|2358087	Keratin compound 2, alkalescence, gene 8[mouse]	70	gi\|13624315
	Trypsinogen 10[mouse]	62	gi\|2358087	Putative protein matter LOC496627[torrid zone Xenopus laevis]	62	gi\|58332100
	Transcriptional regulator LytR family [Dipel serotype subspecies (Bacillusthuringiensis serovarisraelensis) ATCC 35646]	58	gi\|75760497		62	gi\|58332100
		58	gi\|75760497	Prediction: similar to keratin 5b [dog class]	57	gi\|73996461
	Merge to serine peptase (α/β hydrolytic enzyme superfamily) [marine bacteria bacterial strain (Prochlorococcus marinusstr.) NATL2A] to the not qualitative zone of the specific N-end of blue-green algae	55	gi\|72002395	Prediction: similar to keratin 5b [dog class]	57	gi\|73996461
		55	gi\|72002395	Prediction: similar to cytokeratin, partly [pheasant]	54	gi\|50795725
D11	Unnamed protein [mouse]	369	gi\|74146433	Prediction: similar to cytokeratin, partly [pheasant]	54	gi\|50795725
D11	Unnamed protein [mouse]	369	gi\|74146433	The trypsase precursor	182	gi\|136429
	Thioredoxin [Escherichia coli]	69	gi\|148071	The trypsase precursor	182	gi\|136429
	Thioredoxin [Escherichia coli]	69	gi\|148071	Lysozyme	63	gi\|229157
E1	Haemoglobin, the β main chain [mouse] of being grown up	639	gi\|31982300	Lysozyme	63	gi\|229157
E1	Haemoglobin, the β main chain [mouse] of being grown up	639	gi\|31982300	Haemoglobin β-1 chain [mouse]	636	gi\|1183932
	Haemoglobin β-1 chain [mouse]	453	gi\|1183933	Haemoglobin β-1 chain [mouse]	636	gi\|1183932
	Haemoglobin β-1 chain [mouse]	453	gi\|1183933	Alpha-globulin [mouse]	285	gi\|49900
	The trypsase precursor	274	gi\|136429	Alpha-globulin [mouse]	285	gi\|49900
	The trypsase precursor	274	gi\|136429	Beta Globulin chain [homo sapiens]	131	gi\|66473265
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	101	gi\|2781269	Beta Globulin chain [homo sapiens]	131	gi\|66473265
		101	gi\|2781269	Trypsinogen 10[mouse]	66	gi\|2358087
E2	Haemoglobin β-1 chain [mouse]	476	gi\|1183932	Trypsinogen 10[mouse]	66	gi\|2358087
E2	Haemoglobin β-1 chain [mouse]	476	gi\|1183932	Haemoglobin, the β main chain [mouse] of being grown up	475	gi\|31982300
	Haemoglobin β-1 chain [mouse]	334	gi\|1183933	Haemoglobin, the β main chain [mouse] of being grown up	475	gi\|31982300
	Haemoglobin β-1 chain [mouse]	334	gi\|1183933	The trypsase precursor	186	gi\|136429
	Alpha-globulin [mouse]	184	gi\|49900	The trypsase precursor	186	gi\|136429

	The recombinant cell platelet factor 4	54	gi\|209286
	The recombinant cell platelet factor 4	54	gi\|209286	E3	Haemoglobin β-1 chain [mouse]	606	gi\|1183932
	Haemoglobin, the β main chain [mouse] of being grown up	590	gi\|31982300	E3	Haemoglobin β-1 chain [mouse]	606	gi\|1183932
	Haemoglobin, the β main chain [mouse] of being grown up	590	gi\|31982300		Haemoglobin β-2 chain [mouse]	474	gi\|1183933
	Alpha-globulin [mouse]	287	gi\|49900		Haemoglobin β-2 chain [mouse]	474	gi\|1183933
	Alpha-globulin [mouse]	287	gi\|49900		The trypsase precursor	279	gi\|136429
	Beta Globulin chain [homo sapiens]	89	gi\|66473265		The trypsase precursor	279	gi\|136429
	Beta Globulin chain [homo sapiens]	89	gi\|66473265		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	66	gi\|2781269
E4	Haemoglobin, the β main chain [mouse] of being grown up	440	gi\|31982300			66	gi\|2781269
E4	Haemoglobin, the β main chain [mouse] of being grown up	440	gi\|31982300		Haemoglobin β-1 chain [mouse]	396	gi\|1183932
	The trypsase precursor	310	gi\|136429		Haemoglobin β-1 chain [mouse]	396	gi\|1183932
	The trypsase precursor	310	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	249	gi\|2781269
	Alpha-globulin [mouse]	106	gi\|49900			249	gi\|2781269
	Alpha-globulin [mouse]	106	gi\|49900		Lysozyme C (1,4-β-N-acetyl muramidase C)	100	gi\|47117006
	Epidermal cell keratin 2[homo sapiens]	75	gi\|181402		Lysozyme C (1,4-β-N-acetyl muramidase C)	100	gi\|47117006
	Epidermal cell keratin 2[homo sapiens]	75	gi\|181402		Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC 10987]	56	gi\|42784428
E5	Haemoglobin β-1 chain [mouse]	477	gi\|1183932			56	gi\|42784428
E5	Haemoglobin β-1 chain [mouse]	477	gi\|1183932		Haemoglobin, the β main chain [mouse] of being grown up	464	gi\|31982300
	Haemoglobin β-2 chain [mouse]	346	gi\|1183933		Haemoglobin, the β main chain [mouse] of being grown up	464	gi\|31982300
	Haemoglobin β-2 chain [mouse]	346	gi\|1183933		Keratin 10[homo sapiens]	300	gi\|40354192
	Alpha-globulin [mouse]	247	gi\|49900		Keratin 10[homo sapiens]	300	gi\|40354192
	Alpha-globulin [mouse]	247	gi\|49900		Keratin 1[homo sapiens]	225	gi\|17318569
	The trypsase precursor	174	gi\|136429		Keratin 1[homo sapiens]	225	gi\|17318569
	The trypsase precursor	174	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	155	gi\|2781269
	Epidermal cell keratin 2[homo sapiens]	132	gi\|181402			155	gi\|2781269
	Epidermal cell keratin 2[homo sapiens]	132	gi\|181402		Beta Globulin chain [homo sapiens]	93	gi\|66473265
	Lysozyme C-3 (1,4-β-N-acetyl muramidase)	74	gi\|126595		Beta Globulin chain [homo sapiens]	93	gi\|66473265
	Lysozyme C-3 (1,4-β-N-acetyl muramidase)	74	gi\|126595		Main surface glycoprotein [Pneumocystis carinii]	59	gi\|3560519
	Keratin 19[homo sapiens]	58	gi\|7594734		Main surface glycoprotein [Pneumocystis carinii]	59	gi\|3560519
	Keratin 19[homo sapiens]	58	gi\|7594734	E6	Keratin 1[homo sapiens]	845	gi\|17318569
	The trypsase precursor	296	gi\|136429	E6	Keratin 1[homo sapiens]	845	gi\|17318569
	The trypsase precursor	296	gi\|136429		Chain E, leech source property tryptase inhibitors trypsase compound	291	gi\|3318722
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	267	gi\|2781269			291	gi\|3318722
		267	gi\|2781269		Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	194	gi\|73996330
	Cytokeratin 9[homo sapiens]	158	gi\|435476			194	gi\|73996330
	Cytokeratin 9[homo sapiens]	158	gi\|435476		Lysozyme C-3 (1,4-β-N-acetyl muramidase)	76	gi\|126595
E7	Type I keratin 16; K16[homo sapiens]	1177	gi\|1195531		Lysozyme C-3 (1,4-β-N-acetyl muramidase)	76	gi\|126595
E7	Type I keratin 16; K16[homo sapiens]	1177	gi\|1195531		Keratin 14[homo sapiens]	887	gi\|17512236
	Apolipoprotein april protein [mouse]	475	gi\|14789706		Keratin 14[homo sapiens]	887	gi\|17512236
	Apolipoprotein april protein [mouse]	475	gi\|14789706		The trypsase precursor	178	gi\|136429
	Prediction: to keratin 1 isomeride 2 similar [ox]	140	gi\|76617876		The trypsase precursor	178	gi\|136429
	Prediction: to keratin 1 isomeride 2 similar [ox]	140	gi\|76617876		Epidermal cell keratin 2[homo sapiens]	104	gi\|181402
	Mutant keratin 9[homo sapiens]	102	gi\|1890020		Epidermal cell keratin 2[homo sapiens]	104	gi\|181402
	Mutant keratin 9[homo sapiens]	102	gi\|1890020		Lysozyme	87	gi\|229157
	Gelsolin kytoplasm-mouse	68	gi\|90508		Lysozyme	87	gi\|229157
	Gelsolin kytoplasm-mouse	68	gi\|90508	E8	The trypsase precursor	227	gi\|136429
	Unknown [Taylor Lai Shi worm]	207	gi\|82622379	E8	The trypsase precursor	227	gi\|136429
	Unknown [Taylor Lai Shi worm]	207	gi\|82622379		Blood platelet basis protein [mouse]	158	gi\|13560694
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	116	gi\|2781269		Blood platelet basis protein [mouse]	158	gi\|13560694

	Apolipoprotein A-II[mouse]	115	gi\|21618837
	Apolipoprotein A-II[mouse]	115	gi\|21618837		Haemoglobin β	112	gi\|229255
	Apolipoprotein C-I (Apo-CI) (ApoC-I)	67	gi\|114017		Haemoglobin β	112	gi\|229255
	Apolipoprotein C-I (Apo-CI) (ApoC-I)	67	gi\|114017	E9	The trypsase precursor	251	gi\|136429
	Prothrombin [mouse]	160	gi\|15489100	E9	The trypsase precursor	251	gi\|136429
	Prothrombin [mouse]	160	gi\|15489100		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	105	gi\|2781269
	Apolipoprotein A-II[mouse]	59	gi\|21618837			105	gi\|2781269
	Apolipoprotein A-II[mouse]	59	gi\|21618837	E10	Prediction: similar to keratin, type I cytoskeleton 14 (cytokeratin 14) is (CK14) isomeride 3[Bos (K14)	311	gi\|76649703
	Gelsolin kytoplasm-mouse	228	gi\|90508	E10		311	gi\|76649703
	Gelsolin kytoplasm-mouse	228	gi\|90508		The trypsase precursor	113	gi\|136429
	Thioredoxin [Escherichia coli]	81	gi\|148071		The trypsase precursor	113	gi\|136429
	Thioredoxin [Escherichia coli]	81	gi\|148071	E11	The trypsase precursor	180	gi\|136429
	Lysozyme	86	gi\|229157	E11	The trypsase precursor	180	gi\|136429
	Lysozyme	86	gi\|229157		Trypsinogen 10[mouse]	61	gi\|2358087
F1	The trypsase precursor	210	gi\|136429		Trypsinogen 10[mouse]	61	gi\|2358087
F1	The trypsase precursor	210	gi\|136429		Haemoglobin β	136	gi\|229255
	Apolipoprotein C-III[mouse]	129	gi\|15421856		Haemoglobin β	136	gi\|229255
	Apolipoprotein C-III[mouse]	129	gi\|15421856		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	77	gi\|2781269
	The complement component C3 precursor	72	gi\|192392			77	gi\|2781269
	The complement component C3 precursor	72	gi\|192392		Trypsinogen 10[mouse]	64	gi\|2358087
	Apolipoprotein A-II[mouse]	60	gi\|21618837		Trypsinogen 10[mouse]	64	gi\|2358087
	Apolipoprotein A-II[mouse]	60	gi\|21618837	F2	The trypsase precursor	215	gi\|136429
	Apolipoprotein C-III[mouse]	120	gi\|15421856	F2	The trypsase precursor	215	gi\|136429
	Apolipoprotein C-III[mouse]	120	gi\|15421856		The complement component C3 precursor	83	gi\|192392
	Trypsinogen 10[mouse]	68	gi\|2358087		The complement component C3 precursor	83	gi\|192392
	Trypsinogen 10[mouse]	68	gi\|2358087		Apolipoprotein A-II[mouse]	60	gi\|21618837
F3	Keratin 1[homo sapiens]	264	gi\|7331218		Apolipoprotein A-II[mouse]	60	gi\|21618837
F3	Keratin 1[homo sapiens]	264	gi\|7331218		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	247	gi\|2781269
	The trypsase precursor	182	gi\|136429			247	gi\|2781269
	The trypsase precursor	182	gi\|136429		Keratin 10[homo sapiens]	179	gi\|40354192
	Apolipoprotein C-III[mouse]	119	gi\|15421856		Keratin 10[homo sapiens]	179	gi\|40354192
	Apolipoprotein C-III[mouse]	119	gi\|15421856		The complement component C3 precursor	79	gi\|192392
	Apolipoprotein A-II[mouse]	62	gi\|21618837		The complement component C3 precursor	79	gi\|192392
	Apolipoprotein A-II[mouse]	62	gi\|21618837	F4	Lotus E, leech source property tryptase inhibitors trypsase compound	336	gi\|3318722
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	236	gi\|2781269	F4		336	gi\|3318722
		236	gi\|2781269		Apolipoprotein C-III[mouse]	112	gi\|15421856
	Epidermal cell keratin 2[homo sapiens]	72	gi\|181402		Apolipoprotein C-III[mouse]	112	gi\|15421856
	Epidermal cell keratin 2[homo sapiens]	72	gi\|181402		The complement component C3 precursor	69	gi\|192392
F5	The trypsase precursor	258	gi\|136429		The complement component C3 precursor	69	gi\|192392
F5	The trypsase precursor	258	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	148	gi\|2781269
	Apolipoprotein C-III[mouse]	122	gi\|15421856			148	gi\|2781269
	Apolipoprotein C-III[mouse]	122	gi\|15421856		Keratin 1, type II, cytoskeleton-people	117	gi\|7428712
	The complement component C3 precursor	104	gi\|192392		Keratin 1, type II, cytoskeleton-people	117	gi\|7428712
	The complement component C3 precursor	104	gi\|192392		Alpha-globulin [mouse]	69	gi\|49900
	Apolipoprotein A-II[mouse]	63	gi\|21618837		Alpha-globulin [mouse]	69	gi\|49900
	Apolipoprotein A-II[mouse]	63	gi\|21618837	F6	Keratin 1[homo sapiens]	749	gi\|17318569
	Cytokeratin 9[homo sapiens]	443	gi\|435476	F6	Keratin 1[homo sapiens]	749	gi\|17318569
	Cytokeratin 9[homo sapiens]	443	gi\|435476		Keratin 10[rabbit]	277	gi\|87045985
	The trypsase precursor	195	gi\|136429		Keratin 10[rabbit]	277	gi\|87045985

Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	124	gi\|2781269
	124	gi\|2781269		Thioredoxin [Escherichia coli]	112	gi\|148071
Albumin 1[mouse]	69	gi\|29612571		Thioredoxin [Escherichia coli]	112	gi\|148071
Albumin 1[mouse]	69	gi\|29612571		Gelsolin kytoplasm-mouse	58	gi\|90508
Tyrosyl-tRNA synthetase [Lactobacillus saki (Lactobacillus sakei) subsp.sakei 23K]	56	gi\|81428383		Gelsolin kytoplasm-mouse	58	gi\|90508
	56	gi\|81428383	F7	Replenishing C3 precursor (HSE-MSF) [comprising: replenish C3 β chain; Replenish tC3 α chain; C	540	gi\|1352102
Interior alpha keratin inhibitor, heavy chain 4[mouse]	275	gi\|9055252	F7		540	gi\|1352102
Interior alpha keratin inhibitor, heavy chain 4[mouse]	275	gi\|9055252		Cytokeratin 9[homo sapiens]	175	gi\|435476
The trypsase precursor	171	gi\|136429		Cytokeratin 9[homo sapiens]	175	gi\|435476
The trypsase precursor	171	gi\|136429		Prediction: to keratin 1 isomeride 2 similar [ox]	141	gi\|76617876
Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	103	gi\|2781269		Prediction: to keratin 1 isomeride 2 similar [ox]	141	gi\|76617876
	103	gi\|2781269		Apolipoprotein J; SGP-2; TRPM-2[mouse]	94	gi\|6273853
Apolipoprotein A-I precursor-mouse	85	gi\|109571		Apolipoprotein J; SGP-2; TRPM-2[mouse]	94	gi\|6273853
Apolipoprotein A-I precursor-mouse	85	gi\|109571		Trypsinogen 10[mouse]	68	gi\|2358087
Gelsolin kytoplasm-mouse	55	gi\|90508		Trypsinogen 10[mouse]	68	gi\|2358087
Gelsolin kytoplasm-mouse	55	gi\|90508	F8	Keratin 1[homo sapiens]	451	gi\|7331218
Cytokeratin 9[homo sapiens]	387	gi\|435476	F8	Keratin 1[homo sapiens]	451	gi\|7331218
Cytokeratin 9[homo sapiens]	387	gi\|435476		Keratin 10[homo sapiens]	275	gi\|40354192
Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	188	gi\|73996330		Keratin 10[homo sapiens]	275	gi\|40354192
	188	gi\|73996330		The trypsase precursor	175	gi\|136429
Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	105	gi\|2781269		The trypsase precursor	175	gi\|136429
	105	gi\|2781269	F9	Haemoglobin, the β main chain [mouse] of being grown up	317	gi\|31982300
Keratin 1[homo sapiens]	247	gi\|7331218	F9	Haemoglobin, the β main chain [mouse] of being grown up	317	gi\|31982300
Keratin 1[homo sapiens]	247	gi\|7331218		The trypsase precursor	190	gi\|136429
Alpha-globulin [mouse]	186	gi\|49900		The trypsase precursor	190	gi\|136429
Alpha-globulin [mouse]	186	gi\|49900		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	127	gi\|2781269
Trypsinogen 10[mouse]	68	gi\|2358087			127	gi\|2781269
Trypsinogen 10[mouse]	68	gi\|2358087	F10	Keratin 1[homo sapiens]	2235	gi\|7331218
Cytokeratin 9[homo sapiens]	1608	gi\|435476	F10	Keratin 1[homo sapiens]	2235	gi\|7331218
Cytokeratin 9[homo sapiens]	1608	gi\|435476		Type II keratin protein subunit matter	1400	gi\|386854
Prediction: similar to keratin 1; Keratin-1; Cytokeratin 1; Hair alpha protein [chimpanzee]	697	gi\|55638031		Type II keratin protein subunit matter	1400	gi\|386854
	697	gi\|55638031		Keratin 10[homo sapiens]	633	gi\|40354192
Keratin 5[Rattus norvegicus]	469	gi\|33519156		Keratin 10[homo sapiens]	633	gi\|40354192
Keratin 5[Rattus norvegicus]	469	gi\|33519156		Prediction: to keratin 4 isomeride 2 similar [ox]	428	gi\|76617900
Epidermal cell keratin 2[homo sapiens]	394	gi\|181402		Prediction: to keratin 4 isomeride 2 similar [ox]	428	gi\|76617900
Epidermal cell keratin 2[homo sapiens]	394	gi\|181402		Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	376	gi\|73996330
Prediction: similar to keratin, type I cytoskeleton 14 (cytokeratin 14) is (CK14) isomeride 3[Bos (K14)	307	gi\|76649703			376	gi\|73996330
	307	gi\|76649703		The trypsase precursor	264	gi\|136429
Epidermis keratin subunit II	222	gi\|293686		The trypsase precursor	264	gi\|136429
Epidermis keratin subunit II	222	gi\|293686		Lysozyme	94	gi\|229157
Albumin 1[mouse]	87	gi\|29612571		Lysozyme	94	gi\|229157
Albumin 1[mouse]	87	gi\|29612571		Trypsinogen 10[mouse]	68	gi\|2358087
Short-chain dehydrogenase/reductase enzyme SDR[Burkholder belongs to .383]	66	gi\|77965219		Trypsinogen 10[mouse]	68	gi\|2358087
	66	gi\|77965219	F11	Haemoglobin, the β main chain [mouse] of being grown up	495	gi\|31982300
Haemoglobin β-1 chain [mouse]	427	gi\|1183932	F11	Haemoglobin, the β main chain [mouse] of being grown up	495	gi\|31982300
Haemoglobin β-1 chain [mouse]	427	gi\|1183932		Keratin 1[homo sapiens]	333	gi\|7331218

	The trypsase precursor	268	gi\|136429
	The trypsase precursor	268	gi\|136429		Haemoglobin β-2 chain [mouse]	216	gi\|1183933
	Cytokeratin 9[homo sapiens]	184	gi\|435476		Haemoglobin β-2 chain [mouse]	216	gi\|1183933
	Cytokeratin 9[homo sapiens]	184	gi\|435476		Alpha-globulin [mouse]	141	gi\|49900
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	100	gi\|2781269		Alpha-globulin [mouse]	141	gi\|49900
		100	gi\|2781269		Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC10987]	56	gi\|42784428
G1	The trypsase precursor	267	gi\|136429			56	gi\|42784428
G1	The trypsase precursor	267	gi\|136429		Keratin 1[homo sapiens]	198	gi\|7331218
	Apolipoprotein A-II[mouse]	164	gi\|21618837		Keratin 1[homo sapiens]	198	gi\|7331218
	Apolipoprotein A-II[mouse]	164	gi\|21618837		Blood platelet basis protein [mouse]	134	gi\|13560694
	Ubiquitin	121	gi\|223061		Blood platelet basis protein [mouse]	134	gi\|13560694
	Ubiquitin	121	gi\|223061		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	93	gi\|2781269
G2	Keratin 1[homo sapiens]	621	gi\|17318569			93	gi\|2781269
G2	Keratin 1[homo sapiens]	621	gi\|17318569		Keratin 10[homo sapiens]	344	gi\|40354192
	Cytokeratin 9[homo sapiens]	338	gi\|435476		Keratin 10[homo sapiens]	344	gi\|40354192
	Cytokeratin 9[homo sapiens]	338	gi\|435476		Epidermis keratin 2[homo sapiens]	293	gi\|181402
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	270	gi\|2781269		Epidermis keratin 2[homo sapiens]	293	gi\|181402
		270	gi\|2781269		The trypsase precursor	261	gi\|136429
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	179	gi\|73996330		The trypsase precursor	261	gi\|136429
		179	gi\|73996330		Blood platelet basis protein [mouse]	143	gi\|13560694
	Apolipoprotein A-II[mouse]	130	gi\|21618837		Blood platelet basis protein [mouse]	143	gi\|13560694
	Apolipoprotein A-II[mouse]	130	gi\|21618837		Lysozyme C (1,4-β N-acetyl muramidase C)	124	gi\|47117006
	Epidermis keratin subunit II	92	gi\|293686		Lysozyme C (1,4-β N-acetyl muramidase C)	124	gi\|47117006
	Epidermis keratin subunit II	92	gi\|293686		Ubiquitin	66	gi\|223061
	Trypsinogen 10[mouse]	64	gi\|2358087		Ubiquitin	66	gi\|223061
	Trypsinogen 10[mouse]	64	gi\|2358087		Alpha globulin [homo sapiens]	62	gi\|28549
G3	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	197	gi\|2781269		Alpha globulin [homo sapiens]	62	gi\|28549
G3		197	gi\|2781269		The trypsase precursor	173	gi\|136429
	Apolipoprotein A-II[mouse]	147	gi\|21618837		The trypsase precursor	173	gi\|136429
	Apolipoprotein A-II[mouse]	147	gi\|21618837		Blood platelet basis protein [mouse]	139	gi\|13560694
	Ubiquitin	78	gi\|223061		Blood platelet basis protein [mouse]	139	gi\|13560694
	Ubiquitin	78	gi\|223061		Trypsinogen 10[mouse]	66	gi\|2358087
	Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC 10987]	55	gi\|42784428		Trypsinogen 10[mouse]	66	gi\|2358087
		55	gi\|42784428	G4	The trypsase precursor	184	gi\|136429
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	150	gi\|2781269	G4	The trypsase precursor	184	gi\|136429
		150	gi\|2781269		Prediction: to keratin 1 isomeride 2 similar [ox]	139	gi\|76617876
	Blood platelet basis protein [mouse]	128	gi\|13560694		Prediction: to keratin 1 isomeride 2 similar [ox]	139	gi\|76617876
	Blood platelet basis protein [mouse]	128	gi\|13560694		Apolipoprotein A-II[mouse]	96	gi\|21618837
	Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC 10987]	54	gi\|42784428		Apolipoprotein A-II[mouse]	96	gi\|21618837
		54	gi\|42784428	G5	The trypsase precursor	172	gi\|136429
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	147	gi\|2781269	G5	The trypsase precursor	172	gi\|136429
		147	gi\|2781269		Blood platelet basis protein [mouse]	127	gi\|13560694
	Ubiquitin	63	gi\|223061		Blood platelet basis protein [mouse]	127	gi\|13560694
	Ubiquitin	63	gi\|223061		Apolipoprotein A-II[mouse]	60	gi\|21618837
G6	Interior alpha-Keratin inhibitor, heavy chain 4[mouse]	1044	gi\|16741341		Apolipoprotein A-II[mouse]	60	gi\|21618837
G6	Interior alpha-Keratin inhibitor, heavy chain 4[mouse]	1044	gi\|16741341		Keratin 1[homo sapiens]	736	gi\|17318569
	Cytokeratin 9[homo sapiens]	483	gi\|435476		Keratin 1[homo sapiens]	736	gi\|17318569
	Cytokeratin 9[homo sapiens]	483	gi\|435476		The trypsase precursor	253	gi\|136429
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	177	gi\|73996330		The trypsase precursor	253	gi\|136429

	Thioredoxin [Escherichia coli]	114	gi\|148071
	Thioredoxin [Escherichia coli]	114	gi\|148071		Unnamed protein [homo sapiens]	111	gi\|28317
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	107	gi\|2781269		Unnamed protein [homo sapiens]	111	gi\|28317
		107	gi\|2781269		Gelsolin kytoplasm-mouse	74	gi\|90508
	Trypsinogen 10[mouse]	68	gi\|2358087		Gelsolin kytoplasm-mouse	74	gi\|90508
	Trypsinogen 10[mouse]	68	gi\|2358087	G7	Apolipoprotein E	814	gi\|192005
	The trypsase precursor	245	gi\|136429	G7	Apolipoprotein E	814	gi\|192005
	The trypsase precursor	245	gi\|136429		Interior alpha keratin inhibitor, heavy chain 4[mouse]	213	gi\|9055252
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	149	gi\|2781269		Interior alpha keratin inhibitor, heavy chain 4[mouse]	213	gi\|9055252
		149	gi\|2781269		Apolipoprotein A-I precursor-mouse	86	gi\|109571
G8	Chain E, leech source property tryptase inhibitors trypsase compound	390	gi\|3318722		Apolipoprotein A-I precursor-mouse	86	gi\|109571
G8		390	gi\|3318722		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	74	gi\|2781269
	Trypsinogen 10[mouse]	68	gi\|2358087			74	gi\|2781269
	Trypsinogen 10[mouse]	68	gi\|2358087	G9	The trypsase precursor	271	gi\|136429
	Apolipoprotein C-III[mouse]	116	gi\|15421856	G9	The trypsase precursor	271	gi\|136429
	Apolipoprotein C-III[mouse]	116	gi\|15421856		The complement component C3 precursor	80	gi\|192392
	Trypsinogen 10[mouse]	66	gi\|2358087		The complement component C3 precursor	80	gi\|192392
	Trypsinogen 10[mouse]	66	gi\|2358087		Apolipoprotein A-II[mouse]	57	gi\|21618837
	Non-symbiosis haemoglobin [Alnusfirma]	55	gi\|84993584		Apolipoprotein A-II[mouse]	57	gi\|21618837
	Non-symbiosis haemoglobin [Alnusfirma]	55	gi\|84993584	G10	Keratin 6A[homo sapiens]	1409	gi\|15559584
	Keratin 6C[homo sapiens]	1346	gi\|17505189	G10	Keratin 6A[homo sapiens]	1409	gi\|15559584
	Keratin 6C[homo sapiens]	1346	gi\|17505189		Keratin 6B[homo sapiens]	1295	gi\|5031841
	Keratin 6 isomeride K6e[homo sapiens]	1245	gi\|27465517		Keratin 6B[homo sapiens]	1295	gi\|5031841
	Keratin 6 isomeride K6e[homo sapiens]	1245	gi\|27465517		Keratin 6B[homo sapiens]	1240	gi\|21961227
	Keratin 1[homo sapiens]	1182	gi\|17318569		Keratin 6B[homo sapiens]	1240	gi\|21961227
	Keratin 1[homo sapiens]	1182	gi\|17318569		Keratin 10[homo sapiens]	938	gi\|40354192
	Prediction: to keratin 6 IRSs similar [chimpanzee]	765	gi\|55638029		Keratin 10[homo sapiens]	938	gi\|40354192
	Prediction: to keratin 6 IRSs similar [chimpanzee]	765	gi\|55638029		Cytokeratin 9[homo sapiens]	440	gi\|435476
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	387	gi\|73996330		Cytokeratin 9[homo sapiens]	440	gi\|435476
		387	gi\|73996330		The trypsase precursor	180	gi\|136429
	Keratin 19[homo sapiens]	85	gi\|7594734		The trypsase precursor	180	gi\|136429
	Keratin 19[homo sapiens]	85	gi\|7594734		Putative protein matter FG03380.1[Gibberella zeae PH-1]	61	gi\|46115076
G11	The trypsase precursor	219	gi\|136429		Putative protein matter FG03380.1[Gibberella zeae PH-1]	61	gi\|46115076
G11	The trypsase precursor	219	gi\|136429		Apolipoprotein C-III[mouse]	119	gi\|15421856
	The complement component C3 precursor	94	gi\|192392		Apolipoprotein C-III[mouse]	119	gi\|15421856
	The complement component C3 precursor	94	gi\|192392	H1	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	232	gi\|2781269
	The trypsase precursor	183	gi\|136429	H1		232	gi\|2781269
	The trypsase precursor	183	gi\|136429	H2	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	214	gi\|2781269
	The trypsase precursor	188	gi\|136429	H2		214	gi\|2781269
	The trypsase precursor	188	gi\|136429	H3	The trypsase precursor	182	gi\|136429
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	96	gi\|2781269	H3	The trypsase precursor	182	gi\|136429
		96	gi\|2781269		Unnamed protein [black blue spot filefish]	63	gi\|47227197
H4	The trypsase precursor	161	gi\|136429		Unnamed protein [black blue spot filefish]	63	gi\|47227197
H4	The trypsase precursor	161	gi\|136429		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	100	gi\|2781269
	Trypsinogen 10[mouse]	66	gi\|2358087			100	gi\|2781269
	Trypsinogen 10[mouse]	66	gi\|2358087	H5	The trypsase precursor	259	gi\|136429
	Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	148	gi\|2781269	H5	The trypsase precursor	259	gi\|136429
		148	gi\|2781269		Trypsinogen 10[mouse]	62	gi\|2358087
	Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC10987]	56	gi\|42784428		Trypsinogen 10[mouse]	62	gi\|2358087
		56	gi\|42784428	H6	Gsn protein [mouse]	1111	gi\|18606238

	Keratin 1[homo sapiens]	404	gi\|7331218
	Keratin 1[homo sapiens]	404	gi\|7331218		Cytokeratin 9[homo sapiens]	399	gi\|435476
	Interior alpha keratin inhibitor, heavy chain 4[mouse]	399	gi\|9055252		Cytokeratin 9[homo sapiens]	399	gi\|435476
	Interior alpha keratin inhibitor, heavy chain 4[mouse]	399	gi\|9055252		The trypsase precursor	178	gi\|136429
	Thioredoxin [Escherichia coli]	103	gi\|148071		The trypsase precursor	178	gi\|136429
	Thioredoxin [Escherichia coli]	103	gi\|148071		Lysozyme	70	gi\|229157
	Trypsinogen 10[mouse]	64	gi\|2358087		Lysozyme	70	gi\|229157
	Trypsinogen 10[mouse]	64	gi\|2358087	H7	Unnamed protein [mouse]	856	gi\|74146433
	Keratin 1[homo sapiens]	273	gi\|17318569	H7	Unnamed protein [mouse]	856	gi\|74146433
	Keratin 1[homo sapiens]	273	gi\|17318569		Epidermal cell keratin 2[homo sapiens]	269	gi\|181402
	Chain E, leech source property tryptase inhibitors trypsase compound	211	gi\|3318722		Epidermal cell keratin 2[homo sapiens]	269	gi\|181402
		211	gi\|3318722		Keratin 10[rabbit]	156	gi\|87045985
	Thioredoxin [Escherichia coli]	84	gi\|148071		Keratin 10[rabbit]	156	gi\|87045985
	Thioredoxin [Escherichia coli]	84	gi\|148071		Thrombospondin	65	gi\|554390
H8	The trypsase precursor	305	gi\|136429		Thrombospondin	65	gi\|554390
H8	The trypsase precursor	305	gi\|136429		Prothrombin [mouse]	185	gi\|15489100
	Prediction: to keratin 1 isomeride 2 similar [ox]	135	gi\|76617876		Prothrombin [mouse]	185	gi\|15489100
	Prediction: to keratin 1 isomeride 2 similar [ox]	135	gi\|76617876		Analysis with Hen lysozyme stability of spiral dipole and electric charge side chain	127	gi\|2781269
	Apolipoprotein A-I precursor-mouse	107	gi\|109571			127	gi\|2781269
	Apolipoprotein A-I precursor-mouse	107	gi\|109571		Prediction: to trypsinogen 7 isomeride 4 similar [dog class]	96	gi\|73978531
	Trypsinogen 10[mouse]	68	gi\|2358087			96	gi\|73978531
	Trypsinogen 10[mouse]	68	gi\|2358087		Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC 10987]	56	gi\|42784428
H9	Keratin 1[homo sapiens]	426	gi\|7331218			56	gi\|42784428
H9	Keratin 1[homo sapiens]	426	gi\|7331218		Apolipoprotein A-II[mouse]	280	gi\|21618837
	The trypsase precursor	267	gi\|136429		Apolipoprotein A-II[mouse]	280	gi\|21618837
	The trypsase precursor	267	gi\|136429		Cytokeratin 9[homo sapiens]	156	gi\|435476
	Blood platelet basis protein [mouse]	103	gi\|13560694		Cytokeratin 9[homo sapiens]	156	gi\|435476
	Blood platelet basis protein [mouse]	103	gi\|13560694		Trypsinogen 10[mouse]	69	gi\|2358087
H10	Keratin 6A[homo sapiens]	1851	gi\|15559584		Trypsinogen 10[mouse]	69	gi\|2358087
H10	Keratin 6A[homo sapiens]	1851	gi\|15559584		Keratin 6C[homo sapiens]	1802	gi\|17505189
	TPA:TPA_exp: type II keratin K6h[homo sapiens]	1665	gi\|32964837		Keratin 6C[homo sapiens]	1802	gi\|17505189
	TPA:TPA_exp: type II keratin K6h[homo sapiens]	1665	gi\|32964837		Keratin 6 isomeride K6e[homo sapiens]	1650	gi\|27465517
	Keratin 6B[homo sapiens]	1581	gi\|21961227		Keratin 6 isomeride K6e[homo sapiens]	1650	gi\|27465517
	Keratin 6B[homo sapiens]	1581	gi\|21961227		Keratin 10[homo sapiens]	1196	gi\|40354192
	Keratin 1[homo sapiens]	1190	gi\|17318569		Keratin 10[homo sapiens]	1196	gi\|40354192
	Keratin 1[homo sapiens]	1190	gi\|17318569		Keratin 5[homo sapiens]	1076	gi\|18999435
	Type II keratin protein subunit matter	871	gi\|386854		Keratin 5[homo sapiens]	1076	gi\|18999435
	Type II keratin protein subunit matter	871	gi\|386854		Epidermis keratin subunit II	603	gi\|293686
	Prediction: to keratin 6 IRS isomeride 12 similar [dog class]	601	gi\|73996330		Epidermis keratin subunit II	603	gi\|293686
		601	gi\|73996330		Cytokeratin 9[homo sapiens]	575	gi\|435476
	Keratin 3[homo sapiens]	458	gi\|42760012		Cytokeratin 9[homo sapiens]	575	gi\|435476
	Keratin 3[homo sapiens]	458	gi\|42760012		Type II alpha-Keratin IIA[pheasant]	326	gi\|46399073
	Chain E, leech source property tryptase inhibitors trypsase compound	315	gi\|3318722		Type II alpha-Keratin IIA[pheasant]	326	gi\|46399073
		315	gi\|3318722		K15 intermediate filament type I keratin [sheep	279	gi\|3550539
	Type I keratin 10[lung fish]	100	gi\|57335414		K15 intermediate filament type I keratin [sheep	279	gi\|3550539
	Type I keratin 10[lung fish]	100	gi\|57335414		Prediction: to trypsinogen 7 isomeride 4 similar [dog class]	97	gi\|73978531
	Keratin 19[homo sapiens]	91	gi\|7594734			97	gi\|73978531
	Keratin 19[homo sapiens]	91	gi\|7594734		Trypsinogen 7[mouse]	74	gi\|2358072
	Putative protein matter FG03380.1[Gibberella zeae PH-1]	61	gi\|46115076		Trypsinogen 7[mouse]	74	gi\|2358072
	Putative protein matter FG03380.1[Gibberella zeae PH-1]	61	gi\|46115076		Film is in conjunction with transcriptional regulator LytR[wax shape bacillus group ATCC 10987]	56	gi\|42784428

H11	The trypsase precursor	85	gi\|136429
H11	The trypsase precursor	85	gi\|136429	Blood platelet basis protein [mouse]	80	gi\|13560694
	Possible glycosyl transferase [Synechococcus WH 8102]	65	gi\|33632163	Blood platelet basis protein [mouse]	80	gi\|13560694
	Possible glycosyl transferase [Synechococcus WH 8102]	65	gi\|33632163	Keratin 10[homo sapiens]	57	gi\|40354192
	Apolipoprotein A-II[mouse]	56	gi\|21618837	Keratin 10[homo sapiens]	57	gi\|40354192

Table 6

Swiss-Prot marks immediately

Sample protein matter ID score value mark is known

B9 adenylate kinase [clonorchis sinensis] 59 gi|22652628 2

B7 albumin 1[mouse] 1536 gi|29612571 3

A5 alpha globulin [homo sapiens] 147 gi|28549 4

C7 α-Jia Taidanbai 61 gi|191765 2

C8 alpha-globulin [mouse] 321 gi|49900 16

Has the Hen lysozyme stability of spiral dipole and electric charge side chain

G2 analyzes 270 gi|2781269 59

E7 Apoa4 albumen [mouse] 475 gi|14789706 1

A8 apolipoprotein A-I precursor-mouse 112 gi|109571 5

H9 apolipoprotein A-II[mouse] 280 gi|21618837 22

C9 apolipoprotein C2[mouse] 67 gi|817943 1

E8 apolipoprotein C-I (Apo-CI) is 67 gi|114017 2 (ApoC-I)

F1 apolipoprotein C-III[mouse] 129 gi|15421856 15

G7 apolipoprotein E 814 gi|192005 2

F7 apolipoprotein J; SGP-2; The TRPM-2[mouse] 94 gi|6273853 1

C8 beta Globulin [white collar actor monkey] 124 gi|33415435 1

C8 beta Globulin chain [homo sapiens] 134 gi|66473265 6

C8 β-1-globulin [mouse] 432 gi|4760586 1

B12 calmodulin-analog 5[homo sapiens] 195 gi|55859601 1

F4 chain E, leech source property tryptase inhibitors trypsase compound 336 gi|3318722 10

H8 prothrombin [mouse] 185 gi|15489100 2

Replenish C3 precursor (HSE-MSF) and [comprise s: replenish C3 β chain;

F7 replenishes C3 α chain; C 540 gi|1352102 3

F5 complementary element C3 precursor 104 gi|192392 15

Countertrypin=myosin type trypsin inhibitor [mouse, blood plasma,

The D7 peptide moiety, 20aa, 4 fragment 4] 73 gi|407619 2

B8 Guang PROTEIN C precursor [mouse] 98 gi|11762010 1

F10 cytokeratin 9[homo sapiens] 1608 gi|435476 27

The cytokeratin 2[homo sapiens of B9 epidermis] 700 gi|181402 12

Keratin subunit II 603 gi|293686 6 of H10 epidermis

D7 myosin [mouse] 60 gi|2546995 1

57 gi|83719123 1 of B8 Flp pili assembly protein CpaB family [Burkholderia belongs to E264]

E10 gelsolin kytoplasm-mouse 228 gi|90508 6

B12 glyceraldehyde 3-phosphate dehydro-genase [homo sapiens] 62 gi|31645 1

H6 Gsn protein [mouse] 1111 gi|18606238 1

C8 haemoglobin β-2 chain [mouse] 493 gi|1183933 11

E1 haemoglobin β-1 chain [mouse] 636 gi|1183932 14

C8 hemoglobin alpha subunit (hemoglobin alpha-chain) (alpha-globulin) 96 gi|122405 1

C8 hemoglobin alpha subunit (hemoglobin alpha-chain) (alpha-globulin) 172 gi|122474 1

F1 haemoglobin β 136 gi|229255 2

A1 haemoglobin β 182 gi|229301 1

A4 haemoglobin β subunit (HBB) (betaglobulin) 58 gi|122606 1

A3 haemoglobin β subunit (HBB) (betaglobulin) 165 gi|122643 2

The E1 haemoglobin, β adult main chain [mouse] 639 gi|31982300 10

A7 is rich in histidine glycoprotein [mouse] 111 gi|11066003 1

H10 putative protein matter FG03380.1[Gibberella zeae PH-1] 61 gi|46115076 3

D1 putative protein matter LOC338797[homo sapiens] 55 gi|70673359 1

D10 putative protein matter LOC496627[torrid zone Xenopus laevis] 62 gi|58332100 1

D10 putative protein matter LOC77055[mouse] 98 gi|85701680 1

B7 putative protein matter SAV6338[Avid kyowamycin MA-4680] 62 gi|29832880 1

Alpha-Keratin inhibitor in the G6, heavy chain 4[mouse] 1044 gi|16741341 1

Alpha-Keratin inhibitor in the D10, heavy chain 4[mouse] 400 gi|9055252 6

Alpha-Keratin inhibitor in the D10, heavy chain [mouse] 116 gi|9506819 1

H10 K15 intermediate filament type I keratin [sheep] 279 gi|3550539 1

B12 keratin 437 gi|386848 1

B7 keratin 1[homo sapiens] 1518 gi|17318569 21

F10 keratin 1[homo sapiens] 2235 gi|7331218 18

B6 keratin 1, type II, cytoskeleton-people 142 gi|7428712 4

H10 keratin 10[homo sapiens] 1196 gi|40354192 18

C2 keratin 10[rabbit] 288 gi|87045985 4

A11 keratin 14[homo sapiens] 1134 gi|17512236 2

B11 keratin 15 [homo sapiens] 166 gi|30583361 1

B12 keratin 17[homo sapiens] 516 gi|48735384 1

D10 keratin 19[pheasant] 70 gi|45384356 1

H10 keratin 19[homo sapiens] 91 gi|7594734 4

H10 keratin 3[homo sapiens] 458 gi|42760012 1

H10 keratin 5[homo sapiens] 1076 gi|18999435 1

A11 keratin 5[homo sapiens] 349 gi|4557890 1

F10 keratin 5[Rattus norvegicus] 469 gi|33519156 1

D10 keratin 6 IRS 3[homo sapiens] 96 gi|27901522 1

H10 keratin 6 isomeride K6e[homo sapiens] 1650 gi|27465517 2

B10 keratin 6A[homo sapiens] 653 gi|14250682 3

H10 keratin 6A[homo sapiens] 1851 gi|15559584 2

H10 keratin 6B[homo sapiens] 1581 gi|21961227 5

G10 keratin 6B[homo sapiens] 1295 gi|5031841 1

H10 keratin 6C[homo sapiens] 1802 gi|17505189 3

D10 keratin 9[dog class] 79 gi|62122767 1

B10 keratin compound 1, acid, gene 14[mouse] 311 gi|21489935 1

D10 keratin compound 2, alkalescence, gene 1[mouse] 165 gi|6678643 1

D10 keratin compound 2, the c of alkalescence, gene 8[mouse] 70 gi|13624315 1

B12 keratin K5 390 gi|386850 3

B12 keratin type 16 283 gi|186685 1

A11 keratin type II 492 gi|386849 1

Keratin, type II cytoskeleton 4 (cytokeratin-4) (CK-4)

D10 (keratin-4) is (cytoskeleton 57kDa keratin) 135 gi|82654948 1 (K4)

D10 larval keratin XLK[Africa xenopus] 141 gi|13111394 1

D10 LOC495267 protein [Africa xenopus] 98 gi|54261576 1

C2 lysozyme 99 gi|229157 15

B8 lysozyme 73 gi|841217 1

G2 lysozyme C (1,4-β-N-acetyl muramidase C) 124 gi|47117006 3

E6 lysozyme C-3 (1,4-β-N-acetyl muramidase) 76 gi|126595 2

The main surface glycoprotein of E5 [Pneumocystis carinii] 59 gi|3560519 1

Film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC

H8 10987] 56 gi|42784428 13

E7 mutant keratin 9[homo sapiens] 102 gi|1890020 1

The non-symbiosis haemoglobin of D8 [Alnus firma] 64 gi|84993584 3

D10 pancreas trypsase 1[Rattus norvegicus] 78 gi|6981420 2

B8 parvalbumin [mouse] 77 gi|509139 1

D10 phosphoserine phosphatase [having a liking for bitter ancient bacterium DSM 9790] 79 gi|48431085 1

C2 platelet basic protein matter [mouse] 166 gi|13560694 15

A2 platelet factor 4 [mouse] 87 gi|13560695 1

Glycosyl transferase [Synechococcus WH 8102] 65 gi|33632163 1 that H11 is possible

B8 prediction: to keratin 1 isomeride 2 similar [ox] 243 gi|76617876 5

Prediction: similar to keratin 1; Keratin-1; Cytokeratin 1; Head

F10 sends out alpha protein [chimpanzee] 697 gi|55638031 2

A11 prediction: identical with keratin 15 [ox] 209 gi|61813798 1

B12 prediction: to keratin 17 similar [chimpanzee] 184 gi|55644941 1

D10 prediction: to keratin 24 isomeride 1 similar [ox] 112 gi|76644680 1

D10 prediction: similar to keratin 25A [dog class] 163 gi|73965965 1

F10 prediction: to keratin 4 isomeride 2 similar [ox] 428 gi|76617900 2

D10 prediction: similar to keratin 5b [dog class] 57 gi|73996461 1

G10 prediction: similar [chimpanzee 765 gi|55638029 3 to keratin 6 IRSs

The prediction: with keratin 6 IRS isomeride 12 mutually

H10 is like [dog class] 601 gi|73996330 24

Prediction: with keratin 8, type II cytoskeleton is similar-people [black orangutan

A11 orangutan 90 gi|55638407 1

Prediction: similar to keratin, type I cytoskeleton 14 (cytokeratins

B10 14) (K14) (CK14) isomeride 3[Bos 445 gi|76649703 5

Prediction: similar to keratin, type I cytoskeleton 18 (cytokeratins

D10 18) (K18) (CK18) [ox] 71 gi|76617986 1

Prediction: similar to keratin, type II cytoskeleton 5 (cytokeratins

B8 5)(K5)(CK 5)(58kDa cytokerat 262 gi|73996312 1

Prediction: similar to keratin, type II cytoskeleton 8 (cytokeratins

B10 8) (A in the cytokeratin) [mouse 87 gi|62657929 1

Prediction: similar to keratin, type II cytoskeleton 8 (cytokeratins

B12-8) (CK-8) (Keraton-8) (K8) [homo sapiens] 80 gi|88988823 1

D10 prediction: similar to keratin, [pheasant] 54 gi|50795725 1 partly

B10 prediction: be similar to trypsinogen 7 isomeride 4[dog classes 108 gi|73978531 4

B8 Profilin 1[mouse] 111 gi|56206029 1

E2 recombinant cell platelet factor 4 54 gi|209286 1

Merge to not qualitative zone silk ammonia to the specific N-end of blue-green algae

Acid peptidase (α/β hydrolytic enzyme superfamily) [marine bacteria bacterial strain

D10 (Prochlorococcus marinus str.)NATL2A] 55 gi|72002395 1

F10 weak point-chain dehydrogenase/reductase enzyme SDR[bulkholderia cepasea 383] 66 gi|77965219 3

D10 thioredoxin [Escherichia coli] 141 gi|148071 12

D10 thioredoxin [vibrio bacterium ES114] 90 gi|59478765 1

Thioredoxin 1 (TRX1) is [luminous bacillus subsp. (TRX)

D10 laumondii TTO1] 100 gi|36787919 1

Three-D space structure-the S2 of escherichia coli thioredoxin, 2.8 dust branches

D10 distinguishes rate 133 gi|230335 1

H7 thrombostondin 65 gi|554390 1

D10 TPA:TPA_exp: keratin Kb40[mouse] 125 gi|46485130 1

H10 TPA:TPA_exp: type II keratin K6h[homo sapiens] 1665 gi|32964837 1

D10 TPA:TPA_exp: type II keratin Kb36[mouse] 103 gi|46485128 1

Transcriptional regulator, LytR family [Dipel serotype subspecies

D10 ATCC35646] 58 gi|75760497 1

A7 transferrins [mouse] 304 gi|17046471 1

B8 transthyretin [mouse] 95 gi|56541070 1

C6 trypsase (EC3.4.21.4) precursor-Niu 88 gi|67549 1

E4 trypsase precursor 310 gi|136429 79

A3 trypsinogen 10[mouse] 71 gi|2358087 41

H10 trypsinogen 7[mouse] 74 gi|2358072 6

H10 type I keratin 10[lung fish] 100 gi|57335414 4

D10 type I keratin 15 [lung fish] 102 gi|57335394 1

A11 type I keratin 16; K16[homo sapiens] 1301 gi|1195531 4

H10 type IIalpha-keratin IIA[pheasant] 326 gi|46399073 1

B10 type IIalpha-keratin IIB[pheasant] 89 gi|46399075 1

C7 type II keratin Kb1[Rattus norvegicus] 319 gi|57012354 2

D10 type II keratin Kb18[Rattus norvegicus] 182 gi|57012352 1

F10 type II keratin protein subunit matter 1400 gi|386854 2

F6 tyrosyl-tRNA synthetase [Lactobacillus saki subsp.sakei23K] 56 gi|81428383 1

G1 ubiquitin 121 gi|223061 8

D4 the unknown (protein, MGC:116262) [Rattus norvegicus] 190 gi|71051822 1

E8 the unknown [Taylor Lai Shi worm] 207 gi|82622379 1

G6 agnoprotein matter product [homo sapiens] 111 gi|28317 1

C8 agnoprotein matter product [mouse] 352 gi|12845853 1

The unnamed protein of D10 [mouse] 109 gi|26324736 1

The unnamed protein of H7 [mouse] 856 gi|74146433 2

Agnoprotein matter product [paddy rice (monocotyledon japonica rice (japonica

D4 cultivar-group, Japan is fine)] 59 gi|34906342 1

H3 agnoprotein matter product [black blue spot filefish] 63 gi|47227197 1

D10 Zgc:92035[zebra fish] 74 gi|49904349 1

D10 Zgc:92061[zebra fish] 90 gi|49902693 1

Total number 707

Keratin or keratin proteins associated matter 225

Keratin or keratin proteins associated matter 145

Lysozyme or lysozyme proteins associated matter 80

The total protein number 257 that does not contain keratin/insulin/lysozyme

Single protein number 154

Keratin or keratin proteins associated matter 64

Keratin or keratin proteins associated matter 7

Lysozyme or lysozyme proteins associated matter 6

The single protein number 77 that does not contain keratin/insulin/lysozyme

Swiss-Prot steps on

Sample protein matter ID score value mark time marking

H9 apolipoprotein A-II[mouse] 280 gi|21618837 22

C8 alpha-globulin [mouse] 321 gi|49900 16

C2 platelet basic protein matter [mouse] 166 gi|13560694 15

F1 apolipoprotein C-III[mouse] 129 gi|15421856 15

F5 complementary element C3 precursor 104 gi|192392 15

E1 haemoglobin β-1 chain [mouse] 636 gi|1183932 14

The H8 film is in conjunction with transcriptional regulator LytR[Bacillus cercus ATCC10987] 56 gi|42784428 13

D10 thioredoxin [Escherichia coli] 141 gi|148071 12

C8 haemoglobin-2 β chain [mouse] 493 gi|1183933 11

The E1 haemoglobin, β adult main chain [mouse] 639 gi|31982300 10

G1 ubiquitin 121 gi|223061 8

Alpha-trypsin inhibitor in the D10, heavy chain 4[mouse] 400 gi|9055252 6

The E10 gelsolin, kytoplasm-mouse 228 gi|90508 6

C8 globulin chain [homo sapiens] 134 gi|66473265 6

A8 apolipoprotein A-I precursor-mouse 112 gi|109571 5

A5 alpha globulin [homo sapiens] 147 gi|28549 4

B7 albumin 1[mouse] 1536 gi|29612571 3

Complement C3 precursor (HSE-MSF) [comprising: complement C3 β chain; Mend

F7 body C3 chain; C 540 gi|1352102 3

F10 weak point-chain dehydrogenase/reductase enzyme SDR[burkholderia 383] 66 gi|77965219 3

The non-symbiosis haemoglobin of D8 [Alnus firma] 64 gi|84993584 3

The red prosperous PH-1 of H10 putative protein matter FG03380.1[maize] 61 gi|46115076 3

The unnamed protein of H7 [mouse] 856 gi|74146433 2

G7 apolipoprotein E 814 gi|192005 2

H8 prothrombin [mouse] 185 gi|15489100 2

A3 haemoglobin β subunit (HBB) (betaglobulin) 165 gi|122643 2

F1 haemoglobin β 136 gi|229255 2

Countertrypin=myosin type trypsin inhibitor [mouse, blood plasma,

The D7 peptide moiety, 20aa, 4 fragment 4] 73 gi|407619 2

E8 apolipoprotein C-I (Apo-CI) is 67 gi|114017 2 (ApoC-I)

C7 α-Jia Taidanbai 61 gi|191765 2

B9 adenylate kinase [clonorchis sinensis] 59 gi|22652628 2

H6 Gsn protein [mouse] 1111 gi|18606238 1

Alpha-trypsin inhibitor in the G6, heavy chain 4[mouse] 1044 gi|16741341 1

E7 Apoa4 protein [mouse] 475 gi|14789706 1

C8 β-1-globulin [mouse] 432 gi|4760586 1

The unnamed protein of C8 [mouse] 352 gi|12845853 1

A7 transferrins [mouse] 304 gi|17046471 1

E8 the unknown [Taylor Lai Shi worm] 207 gi|82622379 1

B12 calmodulin-analog 5[homo sapiens] 195 gi|55859601 1

D4 the unknown (protein, MGC:116262) [Rattus norvegicus] 190 gi|71051822 1

A1 haemoglobin β 182 gi|229301 1

The three-dimensional of escherichia coli thioredoxin-space structure S2,2.8 dusts are differentiated

D10 leads 133 gi|230335 1

C8 beta Globulin [white collar actor monkey] 124 gi|33415435 1

In the D10-α-inhibitor H4 heavy chain [mouse] 116 gi|9506819 1

A7 is rich in glycoprotein [mouse] 111 gi|11066003 1 of histidine

B8 Profilin 1[mouse] 111 gi|56206029 1

The unnamed protein of G6 [homo sapiens] 111 gi|28317 1

The unnamed protein of D10 [mouse] 109 gi|26324736 1

Thioredoxin 1 (TRX1) is [luminous bacillus subsp. (TRX)

D10 laumondii TTO1] 100 gi|36787919 1

B8 Guang PROTEIN C precursor [mouse] 98 gi|11762010 1

D10 putative protein matter LOC77055[mouse] 98 gi|85701680 1

D10 LOC495267 protein [Africa xenopus] 98 gi|54261576 1

C8 hemoglobin alpha subunit (hemoglobin alpha-chain) (alpha globulin) 96 gi|122405 1

B8 transthyretin [mouse] 95 gi|56541070 1

F7 apolipoprotein J; SGP-2; The TRPM-2[mouse] 94 gi|6273853 1

D10 thioredoxin [Fei Shi vibrios ES114] 90 gi|59478765 1

D10 Zgc:92061[zebra fish] 90 gi|49902693 1

A2 platelet factor 4 [mouse] 87 gi|13560695 1

B8 parvalbumin [mouse] 77 gi|509139 1

D10 Zgc:92035[zebra fish] 74 gi|49904349 1

C9 apolipoprotein C2[mouse] 67 gi|817943 1

H7 thrombospondin 65 gi|554390 1

Protein [black blue spot filefish] 63 gi|47227197 1 that H3 is unnamed

B12 glyceraldehyde 3-phosphate dehydro-genase [homo sapiens] 62 gi|31645 1

B7 putative protein matter SAV6338[Avid kyowamycin MA-4680] 62 gi|29832880 1

D7 myosin [mouse] 60 gi|2546995 1

The unnamed protein of D4 [paddy rice (Japan is fine)] 59 gi|34906342 1

The main surface glycoprotein of E5 [Pneumocystis carinii] 59 gi|3560519 1

A4 haemoglobin β subunit (HBB) (betaglobulin) 58 gi|122606 1

Transcriptional regulatory, [the Dipel serotype subspecies ATCC of LytR family

D10 35646] 58 gi|75760497 1

F6 tyrosyl-tRNA synthetase [Lactobacillus saki subsp.sakei 23K] 56 gi|81428383 1

D1 putative protein matter LOC338797[homo sapiens] 55 gi|70673359 1

Merge to not qualitative regional serine to the specific N-end of blue-green algae

Peptase (α/β hydrolytic enzyme superfamily) [marine bacteria bacterial strain

D10 (Prochlorococcus marinus str.)NATL2A] 55 gi|72002395 1

E2 recombinant cell platelet factor 4 54 gi|209286 1

Total number 257

Particular protein number 77

[0232] although aforementionedly refer to particularly preferred embodiment, can think that the present invention is not so confined.For those those of ordinary skill in the art, can do multiple improvement to disclosed embodiment; And such improvement is intended within the scope of the present invention.

[0233] all public publications, patented claim and the patent quoted are in this manual incorporated the whole of them at this, with for referencial use.

Claims

1. the method for classification or separation comprises

(a) sampling, it comprises first component and second component;

(b) provide substrate, it comprises nano-porous materials; With

(c) described nano-porous materials is exposed to described sample,

During wherein said exposure nano-porous materials, retain described first component but do not retain described second component.

2. according to the described method of claim 1, wherein said sample is the sample of biofluid.

3. according to the described method of claim 2, wherein said biofluid is serum, blood plasma, blood, urine, seminal fluid, refining, liquor pleurae, ascites, milk, ight soil or saliva.

4. according to the described method of claim 1, wherein said first component and described second component comprise peptide, antigen, antibody, protein, protein fragments, RNA or DNA.

5. according to the described method of claim 1, the molecular weight of described second component of the molecular weight ratio of wherein said first component is lower.

6. according to the described method of claim 1, wherein said nano-porous materials is a nano-structure porous silicon.

7. according to the described method of claim 1, wherein said nano-porous materials is the nanoporous oxide material.

8. according to the described method of claim 7, wherein said nanoporous oxide is a nano-stephanoporate silicon dioxide.

9. according to the described method of claim 1, wherein said nano-porous materials is the molecule trapped substance.

10. according to the described method of claim 1, wherein said nano-porous materials has the surface of modification.

11. according to the described method of claim 10, wherein said nano-porous materials has electric charge load surface.

12. according to the described method of claim 10, wherein said nano-porous materials has the surface with modified with functional group.

13. according to the described method of claim 1, wherein said substrate is film, wafer, particle or microchip.

14, according to the described method of claim 1, the described substrate that wherein provides comprises the substrate of making by top-down technology.

15. according to the described method of claim 14, wherein said top-down technology is selected from photoetching process, el, X ray etching, deep-UV lithography and nano impression method.

16., further comprise described first component of extraction from described nano-porous materials according to the described method of claim 1.

17. according to the described method of claim 1, further be included in the described exposure, wash described nano-porous materials.

18. according to the described method of claim 1, wherein nano-porous materials adsorbs described first component.

19., further comprise and analyze described first component according to the described method of claim 1.

20. according to the described method of claim 19, the analysis of wherein said analysis for being undertaken by mass spectroscopy, gel electrophoresis, chromatography chromatogram, biologic test or their combination.

21. according to the described method of claim 20, wherein said mass spectroscopy is MALDI-TOF mass spectroscopy, LC/MS mass spectroscopy, ESI-MS mass spectroscopy, tandem mass spectrometry or SELDI mass spectroscopy.

22. the method for an analytic sample comprises

(a) provide described sample;

(b) provide substrate, it comprises nano-porous materials; With

(c) described nano-porous materials is exposed to described sample; With

To partly analyzing by the described sample that described nano-porous materials kept.

23. according to the described method of claim 22, wherein said sample is the sample of biofluid.

24. according to the described method of claim 23, wherein said biofluid is serum, blood plasma, blood, urine, seminal fluid, refining, liquor pleurae, ascites, milk, ight soil or saliva.

25. according to the described method of claim 22, the wherein said part that retains comprises peptide, antigen, antibody, protein, protein fragments, RNA or DNA.

26. according to the described method of claim 22, the wherein said part that retains is for being adsorbed onto the part on the described nano-porous materials.

27. according to the described method of claim 22, wherein said retain the part be the lower molecular weight part of described material.

28. according to the described method of claim 22, wherein said nano-porous materials is a nano-structure porous silicon.

29. according to the described method of claim 22, wherein said nano-porous materials is the nanoporous oxide material.

30.. according to the described method of claim 22, wherein said nano-porous materials has the surface of modification.

31. according to the described method of claim 22, wherein said substrate is film, wafer, particle or microchip.

32, according to the described method of claim 31, the described substrate that wherein provides comprises the substrate by top-down technology manufacturing.

33. according to the described method of claim 22, wherein said substrate comprises the first area and centers on the second area of described first area that wherein said first area comprises that described nano-porous materials and described second area do not comprise described nano-porous materials.

34. according to the described method of claim 22, the wherein said part that retains has the molecular weight that is no more than 20kDa.

35. according to the described method of claim 34, the wherein said part that retains has the molecular weight that is no more than 15kDa.

36. according to the described method of claim 35, the wherein said part that retains has the molecular weight that is no more than 10kDa.

37. according to the described method of claim 36, the wherein said part that retains has the molecular weight that is no more than 5kDa.

38. according to the described method of claim 37, the wherein said part that retains has the molecular weight that is no more than 4kDa.

39. according to the described method of claim 22, wherein said analysis comprises from the described part that retains of described nano-porous materials extraction.

40. according to the described method of claim 22, the analysis of wherein said analysis for being undertaken by mass spectroscopy, gel electrophoresis, chromatography chromatogram, biologic test or their combination.

41. according to the described method of claim 40, wherein said mass spectroscopy is MALDI-TOF mass spectroscopy, LC/MS mass spectroscopy, ESI-MS mass spectroscopy, tandem mass spectrometry or SELDI mass spectroscopy.

42. according to the described method of claim 22, the low-molecular-weight limit of the detection that wherein said analysis has is at least 20ng/ml.

43., wherein analyze the low-molecular-weight limit of detection that has and be at least 5ng/ml according to the described method of claim 22.

44. be used to detect the method for the sign of physiological condition, comprise

(a) provide the sample that influenced by described physiological condition;

(b) provide the substrate that comprises nano-porous materials;

(c) described nano-porous materials is exposed to described sample;

(d) analyze described sample part by described nano-porous materials kept; With

(e) result and the analysis of control sample gained result with described analysis compares, to detect the sign of described physiological condition.

45. according to the described method of claim 44, wherein said physiological condition is the stage of disease or disease.

46. according to the described method of claim 45, wherein said disease is a cancer.

47. according to the described method of claim 44, wherein said sample is the sample of biofluid.

48. according to the described method of claim 44, wherein said sample is from mammiferous sample.

49. according to the described method of claim 44, wherein said sample is the sample from the mankind.

50. according to the described method of claim 44, wherein said substrate is film, wafer, particle or microchip.

51. according to the described method of claim 44, wherein said nano-porous materials is nanoporous oxide or nano-structure porous silicon.

52. according to the described method of claim 44, the analysis of wherein said analysis for being undertaken by mass spectroscopy, gel electrophoresis, chromatography chromatogram, biologic test or their combination.

53. a kit comprises: be used to collect the instrument of sample, described sample comprises one or more components; And

Substrate, it comprises the nano-porous materials that is set to retain described one or more components.

54. according to the described kit of claim 53, wherein said sample is the sample of biofluid.

55. according to the described kit of claim 53, wherein said porosint is nano-structure porous silicon or nanoporous oxide material.

56. according to the described kit of claim 53, wherein said one or more components are low molecular weight part of described sample.

57. an analytic system comprises

Analytical instrument and

Comprise the substrate of nano-porous materials, wherein said substrate is set to strengthen the sensitivity of analytical instrument to one or more analytes.

58. according to the described analytic system of claim 57, wherein said substrate comprises the first area, described first area comprises nano-porous materials, described one or more analytes can be adsorbed in described first area, described first area is surrounded by second area, and described second area does not adsorb described one or more analytes.

59. according to the described analytic system of claim 58, wherein said second area is non-nanoporous zone.

60. according to the described analytic system of claim 58, the surface of wherein said second area is modified with hydrophilic functional group.

61. according to the described analytic system of claim 58, the surface of wherein said first area is the electric charge load.

62. according to the described analytic system of claim 58, the surface of wherein said first area is modified with functional group.

63. according to the described analytic system of claim 58, wherein said analytical instrument comprises the size of active region of the described ionization source of size match of ionization source and described first area.

64. according to the described analytic system of claim 63, wherein said ionization source is a laser, and the size of the described laser beam of size match of described first area.

65. according to the described analytic system of claim 57, wherein said analytical instrument is a mass spectrometer.

66. according to the described analytic system of claim 65, wherein said mass spectrometer is laser desorption/ionization massspectrum instrument.

67. according to the described analytic system of claim 57, wherein said nano-porous materials is a nano-structure porous silicon.

68. according to the described analytic system of claim 57, wherein said nano-porous materials is a nanometer oxide material.

69. according to the described analytic system of claim 57, wherein said one or more analytes are selected from peptide, antigen, antibody, protein, protein fragments, RNA or DNA.

70. according to the described analytic system of claim 57, wherein said one or more analytes have molecular weight and are no more than 20kDa.

71. according to the described analytic system of claim 57, wherein said one or more analytes have molecular weight and are no more than 15kDa.

72. according to the described analytic system of claim 57, wherein said one or more analytes have molecular weight and are no more than 10kDa.

73. according to the described analytic system of claim 57, wherein said one or more analytes have molecular weight and are no more than 4kDa.

74. a probe comprises

Substrate, it comprises nano-porous materials, and is set to be inserted into in the mass spectrometer.

75. according to the described probe of claim 74, wherein said substrate comprises the first area and centers on the second area of described first area, and wherein said first area comprises nano-porous materials and can retain one or more analytes that described second area does not adsorb described one or more analytes.

76. according to the described probe of claim 75, wherein said second area is non-nanoporous zone.

77. according to the described probe of claim 75, the surface of wherein said second area is modified with hydrophilic functional group.

78. according to the described probe of claim 75, the surface of wherein said first area is the electric charge load.

79. according to the described probe of claim 75, the surface of wherein said first area modified with functional group is to retain described one or more analytes.

80. according to the described probe of claim 75, wherein said mass spectrometer comprises ionization source, and the size of the active region of the described ionization source of size match of described first area.

81. according to the described probe of claim 80, wherein said ionization source is the size of the described laser beam of size match of laser and described first area.

82. according to the described probe of claim 75, wherein said one or more analytes are selected from peptide, antigen, antibody, protein, protein fragments, RNA or DNA.

83. according to the described probe of claim 75, wherein said one or more analytes have molecular weight and are no more than 20kDa.

84. according to the described probe of claim 75, wherein said one or more analytes have molecular weight and are no more than 5kDa.

85. according to the described probe of claim 74, wherein said nano-porous materials is nano-structure porous silicon or nanoporous oxide material.

86. according to the described probe of claim 74, wherein said mass spectrometer is substance assistant laser desorpted ionization massspectrum instrument