CN1890774A - Methods and devices for concentration and purification of analytes for chemical analysis including matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) - Google Patents

Abstract

Analytical methods and devices are disclosed for separating low abundance analytes by electrophoretically driving the analytes through a sieving matrix to first remove high molecular weight species. Subsequently the remaining low abundance analytes are electrophoretically focused onto a capture membrane where the analytes become bound within a small capture site. After this step the capture membrane may be allowed to dry and then attached to a conductive MALDI sample plate.

Description

Enrichment and purifying are used to comprise the method and apparatus of the chemico-analytic analyte that matrix-assisted laser desorption/ionization (MALDI) mass spectrum (MS) is analyzed

Background

1 invention field

This aspect relates to mass spectrum, or rather, relates to the biological sample such as human serum is carried out preconcentration and purifying, thereby carry out substance assistant laser desorpted attached MALDI-MS (MALDI MS) analysis.

2. background of related

With substance assistant laser desorpted attached MALDI-MS (MS) sample that is deposited on the MALDI Target Board is analyzed, become the method that protein, polypeptide and other biomolecule are analyzed rapidly.The MALDI-MS method is very sensitive method, also is the method that adapts most with biological buffer.In addition, can produce the macromolecular ion of large number of biological efficiently, this technology is particularly conducive to macromolecular analysis in pik mole level (subpicomole).To analyzing, bring following a lot of particularity difficult problems to mass spectral analysis such as the peptide analysis thing in the biological sample crude product of blood, blood plasma or serum.

First difficult problem that need overcome is that a large amount of salt (for example, sodium, potassium, chloride, phosphate and carbonate) are arranged in the biological sample.In common maldi analysis method, anion is especially inhibited to the ionization of polypeptide sample.Cation also exists makes main mass spectra peak division produce a plurality of small peaks, causes producing the problem of the cation adduct spectrum with additional cation adduct quality.In addition, successful MALDI-MS analyzes and also depended on analyte before being injected into mass spectrometer, and analyte and analyte MALDI stroma ground substance form the ability of crystallization effectively.The MALDI stroma ground substance need absorb laser, so that analyte generating gasification and ionization in matrix and the adsorbed sample, thereby analyze.Afterwards, Ionized analyte molecule is entered in the mass spectrometer ion detector by the acceleration of the high electric field of the high voltage generation between mass spectrometer inner anode and the negative electrode.Even when having small amounts of contamination such as salt or glycerine, MALDI matrix also can weaken greatly to desorption and the ionization ability such as analytes such as protein and polypeptide.In addition, high salt concentration has improved the laser intensity threshold value of MALDI-MS needs and the intensity of salt adduct polypeptide (under the situation of the free polypeptide signal of infringement).

The second, in sample, compare with disturbing albumen (for example albumin, immunoglobulin (Ig) and transferrins) such as human serum, normally the copy number of analyte polypeptide (copy number) is very low.Target polypeptides usually has only 1 micromoles per liter-1 picomole/liter (for example 1 mg/ml is to 1 pg/ml).In contrast, total albumin and gamma Globulin, as IgG, the horizontal extent of IgM to 0.1 grams per milliliter, is 1 * 10 from 0.01 grams per milliliter ¹¹Quantity doubly.Therefore, main abundant protein takes advantage in the MALDI spectral mixture.Main peaks makes low intensity peak centered faint, thus poor composition difficulty be found.This difficult problem is very serious in such as the biological sample of human serum, exceed the interference albumen (for example albumin, immunoglobulin (Ig) and transferrins) of a plurality of orders of magnitude and salt (for example because exist in the biological sample, sodium, potassium, chloride, phosphate and carbonate), make the detection of low copy number molecule become very difficult.

The 3rd, many analyte polypeptide are hydrophobic, and combine with major protein in blood, blood plasma or the serum, and especially albumin tends to and the hydrophobic molecule non-specific binding.Therefore, remove the loss that unwanted albumen may cause the analyte polypeptide.Such as chemical cracking agent such as salt and cleaning agents, help analyte polypeptide and albuminous dissociating, but these reagent suppress the MALDI process.For example polyethylene glycol (PEG) and Trition efficiently dissociate MALDI, and produce than protein, MALDI signal that polypeptide is stronger.Therefore, these reagent have suppressed the MS signal of protein, polypeptide.After the agent of adding chemical cracking is dissociated polypeptide and albumin, must the albumin that chemical cracking produces be separated with the analyte polypeptide with other contaminating protein.In addition, the separation method that use poor analyte polypeptide in separation process, not lose.When the analyte polypeptide is hydrophobic, when tending to combine, separate very difficulty with hydrophobic surface.If cause the loss of 30% sample with liquid-phase chromatography method purifying biological polymer regular meeting, and can further introduce pollutant.For most of MALDI-MS users, such sample loss is unacceptable.

At last, because the content of analyte polypeptide seldom, carrying out needing enrichment before MALDI-MS analyzes.At first carry out dissociating of polypeptide, the separation of composition, enrichment then.The step that the method that prior art adopts is required great effort and need much be taken time and effort very much.An object of the present invention is to provide the method and apparatus that the easy economized form of a kind of usefulness carries out these steps.Therefore, improve the sample flux, lowered analysis cost.

Reported many technology in the document, carried out the separation of the pollutant before MALDI-MS analyzes with the trouble effort.Usually, the method based on liquid phase or affinity chromatography has obtained using to greatest extent.The separation method that utilizes liquid chromatogram to carry out comprises the connector molecule is connected on the immobile phase immobile phase of functionality (produce) of liquid-phase chromatographic column with chemical method.After sample was packed chromatographic column into, the immobile phase of flowing through mutually flowed.Each analyte has determined the relative migration speed of different analytes (and pollutant and chaff interference) by liquid-phase chromatographic column with the binding time (rather than the time in flowing mutually) of immobile phase, makes analyte obtain purifying.For example, can be attracted on the immobile phase of functionality such as target analytes molecules such as protein and polypeptide, pollutant then flows out from chromatographic column.Then, the target molecule of adjusting on the mutual-assistance immobile phase that flows is isolated.Be applicable to the volatile buffer of MALDI-MS,, can be used as the mobile phase of this step such as acetonitrile/water mixture.Use this mode, the target molecule behind the purifying flows out from the liquid chromatogram pillar, is used for MALDI-MS after the collection and analyzes.Sample does not contain salt, other pollutant that can disturb or weaken sensitivity for analysis to a certain extent at this moment.

Therefore, new device, method and the program that need before MALDI-MS analyzes, carry out enrichment to sample.

Goal of the invention

One of purpose of the present invention provides is carrying out pretreated method to carrying out such as biological samples such as serum, blood plasma, whole blood, cerebrospinal fluid, urines before the sensitive substance assistant laser desorpted attached MALDI-MS analysis.Another purpose provides a kind of easy, high-efficiency method, improves sample flux (throughput), reduces analysis cost.A purpose provides the employed device of a kind of these methods again, the characteristics that this device has is easy to use, quality data is provided, production cost is lower.It would be desirable that this device can provide prerinse and preliminary treatment for the user, and need minimum preparation process and minimum preparation time.Another purpose provides a kind of device, and the production cost of this device is low, can be separately, use economically, thereby in clinical research and people, animal medical clinic applications are used, and needn't consider too much cost and freely use.Because analysis result can not depend on the degree of wear etc. of service condition, cleaning step or the analytical equipment in early stage, thisly can improved consistency and reliability have been brought to the user by prewashed device.The convenience of the manual cleaning device that need not require great effort is provided for the user before use.

In addition, we provide the apparatus and method of electrophoresis, are used to improve the preparation that substance assistant laser desorpted attached MALDI-MS is analyzed preceding sample.These apparatus and method are for the user provides that analyte dissociates, electrophoretic separation, enrichment and capture suitable mutually the catching on the layer of MALDI.Advantageously, catching layer is the slide plate that has plate framework, and this framework and removable attachment combination directly are attached on the MALDI sample panel of mass spectral analysis catches.Carriage is incorporated into sample in the mass spectrometer fast, thereby analyte is handled in a kind of mode of automation.Therefore carriage provides high flux, has reduced the analysis cost of sample.These apparatus and method provide the preparing fast with efficiently of sample for chemical analysis, have separated, enrichment and formative improvement system.The present invention is particularly suitable for the mass spectral analysis preparation of biological sample before.In a word, these apparatus and method are general, and embodiment is provided, and can be used to the single charge anions of enrichment or single electric charge cation or multi-charge anion or the cationic peptide of multi-charge, polypeptide, oligopeptides or protein.In addition, this method can be used for charged carbohydrate, glycoprotein, DNA, RNA or other charged metabolism intermediate.

Electric neutrality molecule (being inner uncharged molecule) also can be analyzed with the electrophoretic apparatus and the method for this paper following discloses, a) electric current by porous fixedly mutual-assistance immobile phase have net charge, the counter ion inducing peripheral solvent streams that flows produces the electroosmotic flow (EOF) that electrophoresis is induced this moment, or

B) make neutral analyte separately by introducing charged glue ion (or charged particle).The basic principle of a kind of separation in back (carrying out in capillary usually) is commonly referred to as Micellar Electrokinetic Chromatography (MECC).These methods can be united use with the apparatus and method that are used to analyze the various molecules of not being with net charge disclosed by the invention.Like this neutral molecule comprise those do not have ionizable group, such as the neutral molecule of pH value near molecule, zitterionic species or other type of the polypeptide of isoelectric point or protein molecule.

The present invention can be used for enrichment hydrophobicity or hydrophilic molecule.The result of this enrichment causes perforated membrane to catch analyte in one or more predetermined, discrete site under concentrated effect of electric field, wherein, regulates the porous capture film, makes it and the mass spectrometric sample panel fit of MALDI that is used for analysis of analytes.For the analyte that helps to be caught carries out ionization, before the porous capture film is put the MALDI mass spectrometer into, MALDI matrix is incorporated into discrete site on the film.The enrichment and the acquisition equipment that have the porous capture film use as described below, so that it can be applicable in the large-scale biomolecule preparation of improvement sample before the MALDI mass spectral analysis.

Description of drawings

Fig. 1 is the perspective view of enricher.

Fig. 2 is the vertical view of enricher.

Fig. 3 is the end view of enricher.

Fig. 4 a is the enlarged side view in cylindrical sample pond.

Fig. 4 b is the amplification plan view in cylindrical sample pond.

Fig. 5 is the enlarged drawing with contacted single pond of photoresponse electrode and porous layer.

Fig. 6 single pond that contains analyte sample and perforated membrane view that to be top electrodes link to each other with voltage source with bottom electrode.

Fig. 7 is that MALDI capture film (having analyte to catch the array in site) is fixed on the end view on the sample panel of MALDI, and wherein catching on the film is added with the solvent that contains MALDI matrix in the site.

Fig. 8 is a slide assemblies, and its top members of frame with separating layer has sample cell, embeds on the basal surface to have in the middle boxes parts of catching layer (vertical view).

Fig. 9 is the top members of frame pitching view of Fig. 8 assembly.

Figure 10 is the underframe parts vertical view of Fig. 8 assembly.

Figure 11 has shown another execution mode of the perforated membrane (8) that contacts with electrode (500), has compression layer (510), is used for analyte being caught and being enriched on the selection part (68) of capture film (60).

Figure 12 has shown the vertical view that is used for by the compression layer (510) of electrophoresis abundance zone charge analysis thing, and analyte is enriched on the selection part of capture film by enrichment hole (518).

Figure 13 is the another kind of version of execution mode shown in Figure 11, has increased other compression layer (510) in the bottom of catching layer (60).

Figure 14 is the device with two-dimensional array pond that is used to separate with enrichment of analyte.

Figure 15 is the end view in pond among Figure 14.

Figure 16 is the device that has electrode and bottom electrolysis liquid pool shown in Figure 15.

Figure 17 is the end view of porous capture film 900 shown in Figure 16, and it is made up of the porous polymeric body integral material (monolith material) in the hole array that is cast on another solid polymerization body layer.

Figure 17 A is the vertical view of porous capture film 900 shown in Figure 16, and it is made up of the porous polymeric body integral material in the hole array that is cast on another solid polymerization body layer.

Figure 18 is the end view of pond array.

Figure 18 A is the view in a pond of device shown in Figure 15, has wherein used porous layer.

Figure 19 is the vertical view of special compression layer (600), and isoelectric focusing IPG band (610) is arranged on it, is used for supplying improved separating effect in the prerequisite of catching of carrying out mass spectral analysis.

Figure 20 is the vertical view of non-permeable formation (600), and it has focusing site (660) under IPG band (610), is used for supplying improved separating effect in the prerequisite of catching of carrying out mass spectral analysis.

Figure 21 is the mass spectrogram of ubiquitin oligo peptide sample, and this sample is with the Texas red marker of (0,1,2,3 or 4 molecule), and on the CEA dialysis membrane held back to 500MW of light enrichment (photo-concentrated).Obtain this collection of illustrative plates by following steps: extract oligopeptides from film and insert the matrix solution that contains CHCA, solution is directly put on the stainless steel Target Board, solution is volatilized.

Figure 22 is the mass spectrogram of identical ubiquitin sample shown in Figure 21.Different is, the ubiquitin sample is enriched to 250 by light, and on the PVDF dialysis membrane that 000MW holds back, the MALDI matrix solution that contains CHCA is directly put on the pvdf membrane, volatilizes, and uses two-sided tape (3M) to paste on the stainless steel MALDI sample panel then.

Figure 23 and 23A are enriched to the mass spectrogram of the electronegative haemocyanin of (using sinapic acid as MALDI matrix) on the PVDF capture film by electricity.

Figure 24 is enriched to the mass spectrogram of the electronegative haemocyanin of (using CHCA as MALDI matrix) on the PVDF capture film by electricity.

Figure 25 and 25A are enriched to the mass spectrogram of the positively charged haemocyanin of (using sinapic acid as MALDI matrix) on the PVDF capture film by electricity.

Figure 26 is enriched to the mass spectrogram of the positively charged haemocyanin of (using CHCA as MALDI matrix) on the PVDF capture film by electricity.

Figure 27 has shown soldering appliance, perforated membrane and PVDF thin layer.

Figure 28 has shown boring guide plate, welding guide plate and base plate.

Figure 29 has shown the bore region of the capture film of welding.The welding girth is represented with diameter, between 0.5-1mm.

Figure 30 has shown the welding film of the MALDI matrix that is coated with 0.5 μ l.

Figure 31 be 5 fly the mole (femtomole) ACTH (fragment 18-39) a mass spectrogram, wherein ACTH is positioned on the porous capture film that is welded on the solid phase pvdf membrane.

Figure 32 is the schematic diagram with the basic embodiment in the hole of perforated membrane.

Figure 33 A and 33B are that analyte is attached to the fluorescent image of the fluorescence analyte (Tr-ubiquitin) on the capture film with before the solvent extraction sample.Left hand view (33A) is the image at the top (using the side of sample) of film, and right part of flg (33B) is the image of the bottom (using the opposing face of sample face) of film.

Figure 34 A and 34B are with suitable release solvent acetonitrile: the analyte after water (50: 50) discharges is attached to the fluorescent image of fluorescence analyte on the PVDF capture film (Tr-ubiquitin).Left hand view (34A) is the image at the top (using the side of sample) of film, and right part of flg (34B) is the image of the bottom (using the opposing face of sample face) of film.

Figure 35 is that the diameter that forms through boring on the condensate supporting layer is that the monoblock porous of 200 μ m is caught the site.

Figure 36 A, 36B, 36C and 36D directly come from the mass spectrogram that 4 on the supporting layer whole porous of separating are caught the site.All detect the ACTH fragment on 4 sites.

Detailed description of the present invention

One aspect of the present invention relates to that a kind of Multi-example analyte to the electrically charged analyte assembled in the electric field separates, enrichment and the apparatus and method of catching. These apparatus and method comprise one or more aspects that the following describes and/or step.

A. store the pond of Multi-example

Fig. 1,2 and 3 have provided respectively perspective view, top view, the side view of enricher. This device is the storage system of pond sample more than, is used for preparing simultaneously in mass spectral analysis one or more samples. The basic function that has provided this device among the figure forms. Device 2 has the sample cell 6 that is arranged on the upper surface 4. Although this device can have different external forms, be generally rectangle, as looking from upper surface 4, one side every being of a size of between the 0.5cm-50cm. This device more usually every limit is of a size of rectangle between the 2cm-15cm. The sample cell of this device can be 1-1000,10-100 pond more usually, the feasibility that this depends on the sample flux and the sample automation is added to the equipment of sample cell. The shape of sample cell can have multiple, and such as cylinder, cube, have the long trapezoid body of the cross section of various shapes, shape of cross section is as square, hexagon, pentagon etc. The diameter in pond or width are usually between 1mm and 1cm. The degree of depth is between 1mm and 2cm, and the degree of depth more commonly used is between 2mm and 10mm.

Fig. 4 a and 4b have provided top view, the side view of the sample cell that amplifies. Each sample cell has sidewall 12, open top 14 and bottom surface 16. Sidewall 12 is to be made by the pore-free material that can keep liquid, aqueous sample, and inner surface 20 and outer surface 22 are arranged. When sample cell was stored sample, aqueous specimen contacted with a part of inner surface 20 of sidewall 12 at least. Aqueous specimen is generally from biological samples such as blood, blood plasma, serum, cerebrospinal fluid, urine, cell extract. Can the storing liquid sample and makes it to rest in the pond in analytic process in the pond. The pond be the storing liquid sample purified with separate (and enrichment) and remove the non-analyte wherein may exist and the analyte that obtains. Usually, the height of sidewall 12 can be stored 1 μ l (microlitre) to the fluid sample of 3ml (milliliter) volume between 1mm and 1cm. For the ease of operating the sample of greater or lesser volume, if required, this device can zoom in or out in proportion accordingly, the size that makes side and bottom surface at 0.1mm between the 10cm. Sidewall 12 and the upper surface 4 of this device are made by pore-free material usually. Pore-free material can be pottery or metal, such as stainless steel, anodized aluminium, copper etc. Usually pore-free material can be following polymer, such as Merlon, and polyethylene, polypropylene, polystyrene, polyimides, nylon, artificial silk, fluorocarbons, perfluocarbon, dimethyl silicone polymer, polyester, acrylic acid (ester) resinoid, acrylonitrile-butadiene-styrene (ABS), polyformaldehyde, polyarylate, polyvinyl chloride, PBT-polyester, polybenzimidazoles acetal copolymer (polybenzimidazone acetal copolymers) acetal copolymer, polyimides, ethene-chlorotrifluoroethylene, the PET polyester, ethene-tetrafluoroethene, PEP, polyethylene, polyurethane, polyketone, polychlorotrifluoroethylene, PET polyester, PPOX, polypropylene styrene, polyether-ether-ketone, polyether sulphone, polyamidoimide, polyarylate, polymethylpentene, polyketone, polysulfones, the mixture of PBT-polyester and/or polymer. Other material also can be used to make enricher, and these materials comprise acrylic acid (ester) resinoid, such as LUCITE^Or Plexiglas; Acrylonitrile-butadiene-styrene (ABS) (ABS); Polyformaldehyde (acetal), polyarylate (ARDEL^); Polyvinyl chloride (PVC); PBT-polyester (CELANEX^); Polybenzimidazoles (Celazole^); Acetal copolymer Celcon or Delrin^ Polyimides is such as Duratron^Or Kaptone^ Ethene-chlorotrifluoroethylene is such as Halar^ The PET-polyester is such as Ertalyte^, ethylene-tetrafluoroethylene is such as Tefzel^ PEP (FEP); Polyethylene; Polyurethane is such as Isoplast^ Polyketone is such as Kadel^ Polychlorotrifluoroethylene (Kel-F^); Polyvinylidene fluoride (PVDF); Poly-terephthalic acids second diester polyester is such as Mylar^ PPOX and styrene are such as Noryl^ Polyether-ether-ketone is such as PEEK^TM Polytetrafluoroethylene (PTFE) (Teflon^); Polyether sulphone is such as Radel^ Polyamidoimide is such as Torlon^ Polyphenylene sulfide is such as Techtron^Or Ryton^ Polyarylate is such as Ardel^ Polymethylpentene (TPX^); Polyketone is such as Kadel^ Polysulfones is such as Udel^ The PBT-polyester is such as Valox^。

B. for separating of the separating layer of analyte and chaff interference

In sample cell or below be two-layer or the porous layer of multilayer 8, be used for separation in the mass spectral analysis of sensitivity, enrichment, reservation and bound analyte.Fig. 5 is two-layer or the amplification cross-sectional view of the exemplary construction of the porous layer of multilayer 8.The bottom surface in pond can all or part ofly be a porous, and all or part of porous layer 8 that is exposed to.When the bottom surface, pond only partly was porous, the bottom surface, pond generally included porous region 24 and imperforate section 26.When being used as such as the structure that improves layer 8 strength of materials or forming hermetically-sealed construction between sidewall 12 and porous layer 8, non-porous district is favourable.

The first porous absorbed layer 30 normally absorbs the liquid absorption layer that is added to the fluid sample in the pond 6.For example, fluid sample can be added in the pond with pipette or other sample dispensing tool, is absorbed into then in the layer 30.Afterwards, the conducting liquid electrolyte that will be used to cushion not significantly under the situation of dilute sample, is added on the absorbed sample.The sample that is not significantly diluted is in close proximity to porous layer 8.Absorbed layer 30 normally is insoluble to the polymer fiber material or the particulate material of the suction of water-based solvent, as cotton or glass fibre, paper or synthetic fibers or particulate.Fiber or particulate can be prepared into by following multiple material, as cellulose, nitrocellulose, cellulose esters, glass fibre, nylon, artificial silk, fluorocarbon, perfluocarbon, dimethyl silicone polymer, polyester, acrylic acid (ester) resinoid, acrylonitrile-butadiene-styrene (ABS); Polyformaldehyde; Polyarylate, polyvinyl chloride, PBT-polyester, polybenzimidazoles acetal copolymer, polyimides, ethene-chlorotrifluoroethylene, PET polyester, ethylene-tetrafluoroethylene, the ethylene, propylene of fluoridizing, polyethylene, polyurethane, polyketone, polychlorotrifluoroethylene, polyethylene terephthalate etc.The major requirement of layer 30 is a) can absorb aqueous specimen, is porous for target analytes.Preferred layer 30 is to be made by Sephadex particle (Pharmacia-Amersham).For example, this Sephadex particle can be G-50-Course, is retained in layer 32 upper surface by fine nylon wire, and nylon wire has the mesh (promptly the diameter 100-300 micron than Sephadex particle is little) of 20 to 100 micron diameters.The volume of Sephadex volume size is per sample regulated.Usually the volume size is between 5 to 100 microlitres.

Chromatographic isolation layer 40 comprises second porous layer.In service at device, layer 40 has different holes for the analyte in being added to pond 6.These analytes can be protein, polypeptide or peptide, and they are according to the difference of molecular size, the difference of electric charge, hydrophobic difference or to the difference of the compatibility of separating layer 40, with different speed by layer 40.Therefore analyte is from pond 6, by separating layer 40, enters the following layer 60 of catching with different speed.Usually, layer 40 comprises one or more separating mediums, and high-quality (being molecular weight) sample component and low-molecular-weight sample component are separated.At this moment, compare the low-molecular-weight sample component, the speed of high-quality sample component by layer 40 time is postponed.For example, layer 40 can be made of the molecular sieve such as dialysis membrane.The technical staff that this dialysis membrane is familiar with clinical dialysis and protein purification to this area knows.Common this dialysis membrane can be made of following material, as cellulose, and cellulose acetate, polyester etc.A problem using this film is if behind the big molecule of excessive loading (reservation molecule), this film is contaminated easily.The severe contamination that excessive loading causes can use convection current (flowing) to remove the method prevention of the reservation molecule on film surface.Can utilize methods such as gravity, hydrostatic pressure, magnetic stirring bar to drive convection current.Preferably, the medium of first separating layer of layer 40 should be such as agarose, is more preferably the porous gel shape material of polyacrylamide.Use the method for delay speed those skilled in the art with micromolecule and big molecular separation to being familiar with, (as DNA, RNA, or protein) is placed in the electric field when charged big molecule, and this gel can not produce pollution.This dependence electrophoresis is all known the gel that very large-scale large biological molecule separates.The another one advantage of this gel is that it can be operated a large amount of samples when electrophoretic separation, does not have excessive problem.When knowing this spawn and under the driving of electric field, hmw protein, DNA, RNA or other biopolymer being filtered, can not pollute and blocking problem by electrophoresis.The known gel of technical staff in albumen and polynucleotides purifying field is polyacrylamide or agarose.Can adjust the filter screen character of this medium by adjusting pore size.For example, the pore size of polyacrylamide can change by the concentration (or concentration of two-crosslinked body of acrylamide) of adjusting acrylamide monomer.

Separating layer 40 can comprise the macromolecular subgrade 52 of a plurality of help separating bios.Second and third, four or more separating medium can be with the combination of first separating medium, continuously or use with mixing.For example, a plurality of subgrades of separating medium can comprise the aperture (in every kind of combination, the aperture increases from the top down successively) that different apertures or size are arranged in gradient, further prevent the obstruction of porous separating layer 40.For example top layer is 2.5% acrylamide, and the intermediate layer is 5% acrylamide, and lower floor is 7.5% acrylamide.In addition, subgrade 52 can by in conjunction with and the specificity substance of removing albumin in whole blood, blood plasma, the serum or IgG form.Specificity to these materials is removed the selective absorption principle that can utilize affinity chromatography.For example, protein, carbohydrate or polynucleotides can be bonded to such as antibody, phytohemagglutin phytolectin or the oligonucleotides above the matrix such as cellulose, dextrin, acrylamide, polymer resin and remove.The method of other selective clearing analyte comprises that with protein and metal (as zinc or nickel) chelating, biotin-avidin effect, hydrophobic dye-albumin bound etc., these technology are known in making up the affinity substrate field.

C. the enriched layer of analyte and catch layer

The 3rd porous enriched layer 50 can choose wantonly be placed on the layer 40 times as enriched layer.Layer 50 provides the material of high mobility to constitute by can be analyte, and this analyte is enriched in the trapping region 68 of catching layer 60.The material that constitutes enriched layer 50 for example is agarose, the cellulose (for example Whatman#1 or Whatman#2 filter paper or analog) of high-permeability.The solid phase of this type of material partly has larger aperture, but prevents that mobile in the enriched layer 50 from remaining favourable.During use, enriched layer 50 is enough thick, prevents that analyte from catching layer 60 from separating layer 40 diffusion and below flowing to.Common thickness is between 200 to 3000 microns.

Enriched layer 50 helps enrichment of analytes (for example concentrate) on the plane parallel with porous layer 8.In case target analytes enter catch the layer 60 before, when passing the separating layer 40 of strong drag, enrichment has just taken place.Though enriched layer 40 is optional existence, when existing, it can help the analyte of enrichment to enter to catch the trapping region 68 in the layer 60.

Below the enriched layer 50 that is positioned at separating layer 40 and chooses wantonly is that analyte is caught layer 60.Catch the layer 60 also be porous so that ionic current pass through.It is enough little usually to catch layer 60 aperture, can make to rely on the capture rate optimization of catching mechanism.Catch mechanism and can be reduced to screening, aperture size is littler than the size of target analytes in such cases.When catch small protein and oligopeptides by screening, the aperture must be quite little, and the order of magnitude is the 10-100 dust.The aperture can less than or greater than target analytes.When catching layer 60 aperture greater than target analytes, catching layer 60 must have affinity to target analytes, as described below.

Catch the normally film of thickness between 1 micron and 1000 microns of layer 60.Thickness more generally is between 10 microns to 200 microns.In thin layer, catch target analytes and be convenient to extract afterwards the analyte that is used for the MALDI-MS analysis.In addition, can make analyte enrichment in proportion with pond dark 18 proportional thin layers 60 of catching.The thickness of catching layer 60 must be enough to make film to have enough mechanical strengths and adhesion.

For a kind of typical separation and catching method, the electric field scope of employing is the 5-100 volt/cm.Common voltage is about 5 volts (being generally about 3 volts to about 10 volts), and the distance between anode and the negative electrode is about 0.4 centimetre (being generally 0.005 centimetre to about 5 centimetres).Electrophoretic velocity is:

V=μ E (formula 1)

μ is the electrophoretic mobility of analyte molecule, and E is an electric field strength.The μ value calculates according to diffusion coefficient D directly by Einstein's relation:

D=(kT/q)) μ (formula 2)

The diffusion coefficient of the molecule of amino acid and similar size approximately is 10 under the room temperature ^-5Cm ²/ second.From place an order 0.0259 volt of numerical value (kt/q) ≈ of electric charge molecule of room temperature, we find that the μ value of these molecules approximately is 4 * 10 ^-4Cm ²/ (volt second).Therefore, according to formula 1, the speed that these molecules pass film in the electric field of about 20 volt/cm is approximately (20 volts/cm) 4 * 10 ^-4Cm ²/ (volt. second)=8 * 10 ^-3Cm/ second (for example about 80 little meter per second).

Our very fast discovery, if the thickness of film is about 80 microns, the time of passing film will be about 1 second.If the thinner thickness of higher electric field, higher electrophoretic mobility or film, then the time of passing will be lacked (for low electric field, low electrophoretic mobility or thicker film, then the time is corresponding elongated) accordingly.Among the present invention, the time of passing film (lacking affinity between analyte and the film) is usually between 0.1 and 100 second.

Catching the mechanism of catching of layer 60 preferably is not only by screening with combining of layer 60 based on analyte.When take place enough in conjunction with the time, then no longer need to catch by screening.When being attached to capture film 60, analyte passes the time of layer 60 and greatly improves (being inversely proportional in the time of passing under the free state with molecule).For example, carry out in conjunction with (0.01% time is free) if molecule on average spends time of 99.99%, then the time of passing is brought up to 104 times.In brief, the time of passing of not carrying out combination is 1 second, and when the binding time of analyte was 99.99%, then the time of passing became about 10,000 seconds.Usually, pass the time of film (under catching layer 60 situation about combining) between 10 to 106 seconds.

By in conjunction with catching and be captured to rare two advantages by screening, a) aperture can be more a lot of greatly than the aperture of screening usefulness, and it is a kind of after catching analyte molecule b) catching layer 60, can be cleaned the film of removing pollutant.When catching with affinity, membrane aperture can be more a lot of greatly than the diameter of the hydrated ion in the biological specimen (as sodium, potassium, calcium and chloride).Catch for carrying out effectively combination, the diffusion length x of film must be only passed in the aperture in passing time t less than analyte.Just as discussed above, t is between 0.1 to 1000 millisecond usually, and d is 10 ^-5To 10 ^-7Cm ²Between/second.Expression formula with three-dimensional diffusion

x ²=6Dt (formula 3)

(diffusion coefficient is approximately 10 to represent small protein ^-6Cm ²/ second) capture rate, or even above-mentioned the fastest by the time (for example 0.1 millisecond), we find that aperture x also may reach 8000 dusts (or almost 1 micron).Contrast, in order to remove molecular weight 1000-60,000 micromolecule analyte, the aperture when catching with screening usually little a lot of times (about 15-40 dust).Therefore, catch for the affinity of electrophoretic velocity very high (or use film), allow to use the aperture from 10 dusts to about 1 micron multiple aperture.Slower electrophoretic velocity or use thick film then to make the corresponding increase in aperture (from 10 dusts to 10 micron or bigger).

Catching like this can be any bonding mechanism.For example contain amino acid whose polypeptide and the protein of being with hydrophobic side chains.So hydrophobic surface and porous hydrophobic membrane tend to suitably high affinity in conjunction with such molecule.This porous film material is as ethylene-tetrafluoroethylene, for example Tefzel ^PEP (FEP); Polyethylene; Polychlorotrifluoroethylene (Kel-F ^); Polyvinylidene fluoride (PVDF); Styrene, for example Noryl ^Polytetrafluoroethylene (Teflon ^) porous Teflon, or similar can be well in conjunction with the material of polypeptide and protein analyte.The PVDF dialysis membrane of preferred molecular weight about 250,000 is (from SpectrumLaboratories; Rancho Dominguez, CA obtains) be used to catch such polypeptide.In addition, by nitrocellulose, nylon, the porous capture film that artificial silk, analogs such as polyester are made has the intrinsic affinity with protein and polypeptide, can be as the capture film of these molecules.In addition, catch layer 60 and can use nitrocellulose, cellulose, nylon, artificial silk, polyester, the film of analogs such as porous PVDF is made.

Catching layer 60 can be the polypeptide binder course, can be by making in conjunction with the material of polypeptide in conjunction with the material of polypeptide or after deriving.Usually, the whole layer 60 of catching is made by same substance.In addition, layer 60 has the polypeptide land or the island of isolation.Thus, analyte can be by enrichment and binder course 60, then with distillation or purify waste water or the buffer solution such as the ammonium carbonate buffer solution for cleaning of predetermined pH and ionic strength.Be combined with the surface of analyte by cleaning, make the impurity that may be present in the sample, be removed as salt or cleaning agent.The material of inhibition or interference analysis thing MALDI signal significantly reduces like this, has improved the sensitivity that analyte molecule is detected.

The layer 60 of catching that is used to catch polypeptide is made by thin, micropore material usually, in this way the dialysis membrane of being made by protein combination material such as polyvinylidene fluoride (PVDF).Layer 60 porous material must be stood water or organic solvent, as aqueous solution buffer solution and electrolyte, methyl alcohol, ethanol, and acetonitrile, these all reagent are by in being usually used in MALDI-MS sample and MALDI matrix.For in conjunction with polypeptide and protein, binder course 60 is normally hydrophobic.Hydrophobicity can be from the organic group of alkyl or aryl, or natural being present in the polymer or by chemical method be connected on the surface, this method is well-known in the art.In addition, hydrophobicity also can be from carbon chlorine compound and fluorocarbon, as ethene-chlorotrifluoroethylene, and perfluocarbon, as polychlorotrifluoroethylene, ethylene-tetrafluoroethylene, PEP, polychlorotrifluoroethylene (Kel-F ^); Polyvinylidene fluoride (PVDF); Polytetrafluoroethylene (Teflon ^) ethylene-tetrafluoroethylene, for example Tefzel ^PEP (FEP) etc.The porosity of dialysis membrane can also allow to rely on molecular size optionally to catch rather than select by hydrophobicity separately.For example the hydrophobicity dialysis membrane of 5,000 molecular weight sizes can be used for the selective retention molecular weight greater than whole molecules of 5,000.With the cleaning agent that contains such as analogs such as octyl glucoside, Triton X-100, NP-40, or clean film such as the elute soln of the organic solvent of analogs such as ethanol, methyl alcohol, acetonitrile, ethyl acetate, the analyte of small-molecular weight wash-out from the film can be gone out.Remove eluate then, protein is attached on the capture film by the hydrophobicity effect.

Form the polymer that the material catch layer 60 also can comprise other and protein bound, as dimethyl silicone polymer, polyester, acrylic acid (ester) resinoid, acrylonitrile-butadiene-styrene (ABS); Polyformaldehyde; Polyarylate, polyvinyl chloride, PBT-polyester, polybenzimidazoles, PET polyester, polyethylene, polyurethane, poly-terephthalic acids second diester polyester, PPOX, polypropylene styrene, polyether-ether-ketone, polyarylether polyarylate, polymethylpentene, PBT polyester, and/or mixture of polymers.Other material that can be used for making enricher comprises that acrylic acid (ester) resinoid is as LUCITE ^Or Plexiglas; Acrylonitrile-butadiene-styrene (ABS) (ABS); Polyformaldehyde (acetal), polyarylate (ARDEL ^); Polyvinyl chloride (PVC); PBT-polyester (CELANEX ^); Polybenzimidazoles (Celazole ^); Acetal copolymer Celcon or Delrin ^Polyimides is as Duratron ^Or Kaptone ^Halar ^The PET-polyester is as Ertalyte ^, polyethylene; Polyurethane is as Isoplast ^Polyketone is as Kadel ^Poly-terephthalic acids second diester polyester is as Mylar ^PPOX and styrene are as Noryl ^Polyether-ether-ketone is as PEEK ^TMPolyether sulphone is as Radel ^Polyamidoimide is as Torlon ^Polyphenylene sulfide is as Techtron ^Polyarylate is as Ardel ^Polymethylpentene (TPX ^); Polyketone is as Kadel ^Polysulfones is as Udel ^Polypropylene sulfide is as Ryton ^The PBT-polyester is as Valox ^The film that mixture of polymers forms, for example Xenoy ^Or the laminate layers of two-layer or multi-layer polymer film.

The porous capture film has the discernible mark position of mass spectrometer, has a precalculated position on this mark position at least, and the distance of itself and mark position is known.The precalculated position is corresponding to trapping region, and trapping region is to catch layer zone of last enrichment target molecule.Mark position can be the black non transparent district.The electrophoretic current that device 2 focuses on preferentially passes through trapping region 68 rather than the zone 62 in the capture film 60, thereby causes selected analyte to obtain enrichment in zone 68.(see that the cross section shown in 66 lines (cross-section) is inversely proportional to, this area is relevant with the cross-sectional area of whole sample cells 6 of capture film 60 tops for the area in enriching quantity and zone 68.Line 64 has shown the cross section in pond.Therefore enriching quantity is directly proportional with square square institute's value divided by line 66 length of line 66 length.The analyte of the enrichment film 60 that is hunted down is caught, so that they were placed on the MALDI Target Board with enriched form (as conc forms) afterwards, thereby can be extracted out from this enriched form.The diameter in example enrichment district is usually between 1 to 1000 micron.The diameter of rich region more generally is between 50 to 200 microns.

Catch layer 60 if analyte passes, can below catching layer 60, place barrier layer 70 spilling with the prevention analyte.Be similar to layer 30,40,50 and 60, layer 70 also is a porous.The aperture of layer 70 is enough little, spills from layer 70 with the target analytes that prevents all selections.Barrier layer 70 can be any dialysis membrane that can hold back small molecular weight material, for example from Spectrum Laboratories, the material of the CEA dialysis membrane energy molecular cut off about 500 that Inc. obtains, itself and PVDF capture film 40 can be cooperated well, are used for keeping the target peptide that human plasma or serum may exist.Any suitable dialysis membrane, or other has the film of suitable aperture, all can be used as the barrier layer.If catch by screening on the upper strata of analyte tegillum 60 61, then need not barrier layer 70.

Similarly, be arranged on that to catch below layer 60 and the barrier layer 70 be the optional resilient coating 80 that exists.Resilient coating can be solid, liquid, perhaps the associating of the two.For example resilient coating can be the liquid solution of the buffering matrix that concentrates, as the histidine aqueous solution of 250mM.The pH value of best buffer solution for histidine, is about 7.5-8.0 near the isoelectric point of required buffer substance.Thereby under the ionic conductivity of minimum, buffer capacity is very strong.Simultaneously, the saturated solution of aspartic acid or glutamic acid, or other zwitterionic buffer also can be used for the similar buffering of isoelectric pH 2.5-3.0 scope.Perhaps, such buffer solution can be blended in the aqueous gel or sol-gel that comprises such as agarose or polyacrylamide.

Porous layer 8 can comprise all each layers or wherein which floor of layer in 30,40,50,60,70 and 80.Separating layer 40 is that device is essential with catching layer 60, and layer 30,50,70 and 80 can be chosen existence wantonly.The layer that these are optional has improved the performance of device, comprises firmer operability, separates fast, catches more fully, or improves the stable of pH or have concurrently.

D. the electrode that is used for focusing electric field

The bottom surface of porous layer 8 is bottom electrodes 100, produces ion flow in the porous layer 8 thereby this electrode can provide electric current to make.Comprise all layers of the layer 8 of layer 30,40,50,60,70 and 80, they are porous to the ion in the electrolyte sample, make electrophoresis attract analyte when electrode 100 motions thus, and ion flow gathers on the zone 68 of catching film 60.

Fig. 6 has provided the schematic diagram in single sample pond 6, and it contains the sample analytes in conductive ion liquid electrolyte 90.

Top electrodes 140 is immersed in the sample electrolyte 90 and is connected with lead 150 as counterelectrode.The other end of lead 150 is connected in power supply 160, and this power supply also is connected in lead 120, and lead 120 also is connected in bottom electrode 100.Power supply applies bias voltage to top electrodes 140, and top electrodes 140 has electromotive force with respect to bottom electrode.Element 6,8,12,90,100,120,140,150 and 160 actings in conjunction are formed with the electrolytic cell of electrophoretic separation function.As with a pressurizer, this bias voltage can be predetermined or steady state value or programming become in time.In addition, power supply can be the constant potential meter, and the electric current that wherein passes electrolytic cell is that be scheduled to or fixing.In a preferred mode of operation, power supply is the constant potential meter, and electrolytic cell can move by potentiostatic mode in the scope between 100 microamperes to 10 milliamperes.More commonly, electric current is between about 400 microamperes to 1 milliampere.After current value is scheduled, the scope of voltage between about 1 to 100 volt.More commonly, voltage is between 2.0 volts and 20 volts.

Preferred bottom electrode is photoconduction (photoconductive) electrode, and it is limited in the photoconduction zone 200 of electrode 100 current path in the electrode.Light-guide material can be the semiconductor such as doped silicon or germanium, GaAs, titanium dioxide, tungsten oxide or analog.These photoresponses (photoresponsive) material can be a monocrystal, or polycrystal, promptly a plurality of small crystalss.In addition, photoconduction electrode 100 can be the film that the amorphous material is made.For example, can obtain such film by evaporation or sputter deposition.Preferred semi-conducting material can be by low-cost screen printing technique depositing Ti O ₂Or similarly semi-conducting material and the thick film made.The method that is used for making the light-guide material of thin or thick film is well known in the prior art.

Use will cause the generation in photoconduction zone 200 from the focusing of light source 180 or parallel light beam 190 irradiates light conductive electrode.Zone 200 comprises the irradiated area of electrode 100 and centers on the diffusion length d of each irradiated area minority carrier.For material such as pure monocrystalline germanium, can be apart from d substantially in 1 millimeter magnitude or bigger.Can be in 100-500 micron number magnitude in pure monocrystalline silicon apart from d, or, make distance less than 100 microns even less than 1 micron by in single crystal semiconductor, mixing the material that can shorten minority carrier lifetime in right amount.Such material or " life-span killer " are the metals such as gold, iron, copper etc. that this area is familiar with.Therefore, size that can basic controlling photoconduction zone 200.The diameter of light beam is usually between 0.001 to 1 millimeter.Utilize little focused light source, directly pass aperture lens or condenser lens such as laser or other light source, the width of light can be a 1-100 micron or still less, produces diameter from 1-500 micron or littler photoconduction zone 200.

Can make the photoresponse electrode with single continuous semi-conducting electrode, to produce many arrays that contain a plurality of electrolytic cells (for example referring to Fig. 1,2 and 3) that two or more photoconduction zones 200 are arranged.Photoresponse electrode 100 can be connected to power supply 160 by one or more lead 120, applies bias voltage with respect to counterelectrode 140 for photoresponse electrode 100.Two or more electrolytic cells are connected with single photoresponse electrode, and the luminous intensity in the photoconduction zone 200 by control directive photoresponse electrode 100 makes the light stream of passing each electrolytic cell controlled respectively.Can control the light stream of passing each electrolytic cell independently by the luminous intensity of control beam 190.For example, under the potentiostatic mode, can be by between electrode 140 and 100, setting in advance single bias voltage, respectively electrolytic cell is operated, but distinguish the light stream of monitoring independently by electrolytic cell, being respectively to each electrolytic cell provides the light source of light beam that feedback signal is provided, and makes the light stream of passing electrolytic cell keep predetermined value.Under the constant potential mode, can operate each electrolytic cell by the control illumination intensity.Power supply is the constant potential meter in a preferred operational mode, and the electric current of selection is in 100 microamperes to 10 milliamperes scopes.More common electric current is between about 400 microamperes to 1 milliampere.After electric current was predetermined, voltage was within the 1-100 volt range.More common voltage is between 2.0 volts and 20 volts.

The top electrodes 140 that serves as counterelectrode needs not to be photoresponse.Top electrodes 140 is preferred by forming such as the insertion material of platinum, gold, palladium etc.Top electrodes 140 also can be used the less expensive material, such as materials such as stainless steel, titanium, chromium.In fact the material of any conduction can be used as top electrodes, as long as those electrode materials are not corroded by aqueous specimen or dissolve.For the attenuating expense keeps optimum corrosion resistance again, top electrodes 140 can be the material that inert material is plated to less expensive, on iron, copper, titanium, tungsten brass etc.Counterelectrode also can be made of carbonaceous material such as carbon, the graphite etc. of conduction.Its advantage is that available screen printing technique places carbonaceous material on the non-conductive condensate, produces conductive materials with cheap manufacture method thus.

The invention provides a quick system, be used for from the complex biological sample that has interfering materials such as salt, lipid and sugar such as blood plasma, serum and cerebrospinal fluid etc., preparing and the analyte that separates needs.Then the analyte of Fen Liing with " Z " dimension (dimension) mode by enrichment, capture on the film 60.By trapping region 68 electric field is accumulated in each sample electrolytic cell, analyte also can promptly be enriched on the trapping region 68 in the plane of capture film 60 with " X " and " Y " dimension.

E. the capture board that has the site array of catching can provide removable load on it to the MALDI Target Board

Thing is used for the MALDI mass spectral analysis

After analyte separates and is enriched in the zone 68 of capture film 60, carry out the MALDI mass spectral analysis, binder course 60 separated with other porous layer, and with binder course 60 attached on the sample panel 300 that is suitable for the MALDI mass spectral analysis, introduce then in the mass spectrometer shown in Figure 7.The suitable MALDI matrix that will be dissolved in then in the suitable matrix solvent is added on the conjunctival capture region 68 with droplet 350.Make the dissolution with solvents analyte, treat solvent evaporation after, on the end face 61 of capture film 60, form the MALDI host crystal mix analyte.In MALDI mass spectral analysis field, well-known matrix is organic acid normally, and the electromagnetic spectrum scope that these organic acids excite at ultraviolet laser (pulse nitrogen laser) (for example 337 nanometers) has strong energy absorption.Usually MALDI matrix adds to dissolve analyte again with liquid form, add such as 0.5 to 5.0 microlitre with small size, in order sample analytes significantly not to be diluted, in order to avoid make it be tiled in a large tracts of land slightly, so that surpass the end face 61 of analyte capture film 60 capture regions 68.Can reduce the volume of matrix by means of the automation distributor, for example 1 skin is raised to 1 and receives liter.More common matrix volume is received between the liter at 1 microlitre and 1.Treat that the solvent liquid evaporation forms after the MALDI host crystal, sample panel is about to be sent to mass spectrometer.After MALDI sample panel 300 was inserted into mass spectrometer, the MALDI host crystal was caused a part of analyte molecule to be ionized by strong UV laser pulses irradiation, and this technology is that this area is well-known.Measure the ion that whether has the discrete molecules amount then, this method also is that this area is well-known.

Sample on 2 pairs of standard sample plates of use device carries out MALDI-MS and analyzes.The MALDI-MS sample panel is made of electric conducting material usually, prevents the electrostatic charging of sample surfaces in the auxiliary ionization process of laser.The cross section of capture film 60 that installs 2 inside is very thin usually, for example 10 microns between 200 micron thickness, electrostatic charge can with MALDI sample panel capacitive coupling.

Charging produces any ill-effect that high pressure prevents electric field thereby capacitive coupling prevents sample surfaces, and this electric field can quicken the ionized sample in the MALDI mass spectrometer.Thin non-conductive device in addition can be made of conductive materials, so that this device has rational thickness.In the present example, the thickness of capture film 60 can be 1mm or thicker.

Conductive materials can be above-mentioned any condensate that adds conductive materials.For example added with the carbon of the conduction of amorphous carbon or graphite or, can greatly increase the conductance of capture film, this is well-known.This device can be by the metal of conductivity, such as formations such as stainless steel, aluminium, yellow gold, silver, palladium, copper, chromium in addition.In other embodiments, semi-conducting material doping or intrinsic also can provide conductance to device.If semiconductor is an intrinsic, the electromagnetic radiation that is higher than the semiconductor band gap can provide sufficient conductance for semiconductor device, eliminates any electric charge on the capture film.

Well-known to the mass spectrometric operating personnel of MALDI is 22 location, small sample pond can be excited 22 surfaces, pond with excitation laser.As shown in following embodiment, comprise the exciting of enrichment of analyte 30 positions of pond base surface area 26, can improve the sensitivity of detection of analytes.Matrix generally is to be put in the acid solution to mix the solubility that improves matrix with organic solvent, and after organic solvent evaporation, matrix is with regard to crystallization.

The example of typical MALDI matrix includes, but are not limited to sinapic acid (C among the present invention ₁₁H ₁₂O ₅), be commonly called alpha-cyano-4-hydroxycinnamic acid (C of CHCA ₁₀H ₇NO ₃), 3,4,5-trimethoxy cinnamic acid, gentianic acid (C ₇H ₆O ₄), trihydroxy-acetophenone C ₈H ₈O ₄), leucoalizarin (C ₁₄H ₁₀O ₃), 2-4-hydroxy phenyl azo group)-benzoic acid (C ₁₃H ₁₀N ₂O ₃), the 2-amino benzoic Acid, trans-3-indyl acrylic acid (C ₁₁H ₉NO ₂), forulic acid (C ₁₀H ₁₀O ₄), nicotinic acid-nitrogen oxide (C ₆H ₅NO ₃), 2 '-6 ' resacetophenone (C ₈H ₈O ₃), picolinic acid (C ₆H ₅NO ₂), the acid of 3-hydroxy-methyl pyridine and 6-azepine-2-thymidine (C ₄H ₅N ₃OS).Usually, these MALDI substrate molecules are dissolved in the organic solvent miscible with water, in acetonitrile, acetone or methyl alcohol.The acidic aqueous solution of solvent and matrix and these molecules mixes mutually as formic acid, acetate or trifluoroacetic acid.Acid wherein guarantees that with so that the pH of MALDI matrix solution keeps sufficient acidity air-dry back forms acidic crystallization rather than salt crystallization.Acid solution guarantees that also polypeptide and protein analyte exist with protonation state, and this moment, the stimulation effect of MALDI was best, and this is well-known in the art.Typical MALDI matrix solution comprises the acetonitrile solution (CHCA or the sinapic acid that comprise 20mg/ml) of 50% (volume) and 50% the 0.1%TFA aqueous solution.

For the ease of device is operated, making can provide the array of two or more sample cells and can provide sealing porous layer 8 and the parts of fast this device of quick-detach, can with keeping sample catch layer 40 and MALDI matrix is convenient, directly insert in the mass spectrometer.Fig. 8 has provided the vertical view of slide assemblies 400, and it has top members of frame 410 and underframe parts 440.After finishing sample separation and analyte and being captured to capture film 40, top members of frame and underframe parts can be disassembled mutually.Top members of frame and underframe parts 440 are beneficial to the dismounting and the installation of catching layer.Top members of frame 410 has constituted the end face 4 of separation and enriching apparatus 2.In end face 4 top castings or get out jag and form sample cell 6.To pour into to form in the pond such as the polymeric liquid that contains acrylamide monomer and crosslinking agent needs thickness, can easily separating layer 40 be inserted pond 6.As by adding sensitising agent such as chain exciting agent such as Ammonium Persulfate 98.5 or riboflavin or allow riboflavin absorb the method for ultraviolet or 400-450nm light, liquid is with regard to polymerization.This method of utilizing polymerization to prepare gel is known for this specialty.

Fig. 9 is the upward view that the top members of frame 410 of bottom surface 414 is shown, and basal surface 414 has from the outstanding downwards projection (projections) 412 of end face 4.The bottom that separating layer 40 is filled each projection covers basal surface 416.By this approach, separating layer can be directly for example be caught layer 60 with porous layer subsequently or optional enriched layer 50 contacts.Members of frame 410 has 0.5 usually to the thickness of 10mm, more common from 0.7 to 2 millimeter.The thickness that the total depth in pond 6 equals members of frame 410 adds that the length of projection 412 deducts the thickness (and the degree of depth that deducts optional protective layer 30) of separating layer 40 in the projection.

Figure 10 demonstrates the vertical view of the underframe parts 450 of skateboard component 400.The end face 452 of frame part 450 has hole 454, extends through members of frame 450 in any direction.Layer 60 (or the enriched layer 50 of letting alone choosing is earlier caught in placement on the basal surface 456 of members of frame 450, be to catch layer 60 then), so that separating layer 40 (being placed in the projection 412 of top members of frame 410) can with catch the layer 60 or enriched layer 50 directly contact the length of top members of frame 410 protrusions 412 and the thickness approximately equal of frame part 450.

In the top 452 of underframe parts 450, extrude a depression 458, depression 458 is ferromagnetic materials, such as the steel disc of about 1 millimeters thick.When the sample on mass spectral analysis film 60 was analyzed, the function of ferromagnetic material was that underframe parts (together with the capture film 60 that adheres to) are fixed firmly on the MALDI sample panel 300.For this reason, the MALDI sample panel has has installed and fixed magnet at corresponding site.

F. be used for improving that electric field is assembled and from catching the device of slide plate chemical extraction analyte

Selectable specific embodiments is seen Figure 11 in one.This scheme and Figure 10 are similar, except in separating layer 40 with catch layer and increased compression layer 510 between 60.Shown in Figure 12, compression layer 510 has impermeability zone 512 and aperture 518, allows to see through impermeable regional 512 to the ionic conduction of catching layer 60 from separating layer 40.The function in hole 518 is to force the only little cross section capture region 68 by capture film 60 of ionic current, the center in hole 518 in fact with the center-aligned of the capture region 68 of capture film 60.The diameter in hole is usually between 10 microns and 2 millimeters.More common diameter is between 50 and 500 microns.Compression layer 510 can be arbitrary thickness, but for reaching lasting and compact purpose, usually between 50 microns and 1000 micron thickness.When especially compressive films thickness is greater than 200 microns, it to the precipitation porous in the hole, suction, hydrophilic material is very useful, does not have bubble with the water buffer solution in the retaining hole.The hydrophobe of suction can be selected from multiple material, for example: agarose, sephadex, emulsion, silicon dioxide microparticle, glass particle etc.The diameter of hydrocolloid particle is littler than the thickness of compression layer 510.The electric charge of careful attention particle surface.For example when positively charged macromolecule analysis thing passed hole 518 by the electrophoresis enrichment, it was positive material that the hydrophobe of suction should be selected neutrality or surperficial net charge.The surface net charge is when negative, only to be used as when the distance of the distance between surface charge during greater than big molecule positively charged.Similarly, when electronegative macromolecule analysis thing passes hole 518 by the electrophoresis enrichment, the hydrophobe of suction will be selected neutral or surperficial net charge is negative material.The surface net charge is timing, only to be used in the distance when the distance between surface charge is electronegative greater than big molecule.

The material that can be used for preparing compression layer 510 comprises metal, as aluminium, titanium, chromium, zinc, tantalum, tungsten, or the mixture of itself and other element.The preferred polymeric material is as Merlon, polyethylene, polypropylene, polystyrene, polyimides, cellulose, nitrocellulose, cellulose esters, nylon, artificial silk, fluorocarbon, perfluocarbon, dimethyl silicone polymer, polyester, acrylic acid (ester) resinoid, acrylonitrile-butadiene-styrene (ABS); Polyformaldehyde; Polyarylate, polyvinyl chloride, PBT-polyester, polybenzimidazoles; Acetal copolymer; Polyimides; Ethene-chlorotrifluoroethylene; The PET-polyester, ethylene-tetrafluoroethylene; PEP; Polyethylene; Polyurethane; Polyketone; Polychlorotrifluoroethylene; Poly-terephthalic acids second diester polyester; PPOX; Polypropylene styrene; Polyether-ether-ketone; Polyether sulphone; Polyamidoimide; Polyarylate; Polymethylpentene; Polyketone; Polysulfones; PBT-polyester and/or mixture of polymers.Other material that can be used for making enricher comprises that acrylic acid (ester) resinoid is as LUCITE ^Or Plexiglas; Acrylonitrile-butadiene-styrene (ABS) (ABS); Polyformaldehyde (acetal), polyarylate (ARDEL ^); Polyvinyl chloride (PVC); PBT-polyester (CELANEX ^); Polybenzimidazoles (Celazole ^); Acetal copolymer Celcon or Delrin ^Polyimides is as Duratron ^Or Kaptone ^Ethene-chlorotrifluoroethylene ethene is as Halar ^The PET-polyester is as Ertalyte ^, ethylene-tetrafluoroethylene is as Tefzel ^PEP (FEP); Polyethylene; Polyurethane is as Isoplast ^Polyketone is as Kadel ^Polychlorotrifluoroethylene (Kel-F ^); Poly-terephthalic acids second diester polyester is as Mylar ^PPOX and styrene are as Noryl ^Polyether-ether-ketone is as PEEK ^TMPolytetrafluoroethylene (Teflon ^); Polyether sulphone is as Radel ^Polyamidoimide is as Torlon ^Polyphenylene sulfide is as Techtron ^Polyarylate is as Ardel ^Polymethylpentene (TPX ^); Polyketone is as Kadel ^Polysulfones is as Udel ^The PBT-polyester is as Valox ^Other pore-free material also can use, but especially preferred material is polyimides (Kapton ^).

The second optional compression layer 520 can be placed on below the capture film 60, and capture film 60 is placed between first and second compression layers, as shown in figure 13 in the mode of " sandwich ".

Impermeable regional 512 of first compression layer 510 can weld together with reserve area, second compression layer impermeable regional 522 of capture film 60, moves (drift in diffusion, convection current or the electric field) zone 62 to capture film 60 to prevent the analyte in capture film 60 capture regions 68.This is caught analyte and will be retained in the zone 68, in follow-up sample preparation steps, for example add can keep after the MALDI matrix more.Can weld film with the means of solvent or heating, the preparation of this polymer and metal material and the technology of welding are well-known.

G. further improve the separating step of detection sensitivity

Apparatus and method of the present invention can with traditional separating and the enriching step combination, reach further raising separation and concentration effect, produce high detection sensitivity.Additional step needs the extra time, but additional step can combine with the step among the present invention and carries out the automation mode and operate.Therefore these steps are easy and simple to handle, require seldom or needn't personnel intervene or personnel participate in.

I) isoelectric focusing:

For example, isoelectric focusing step can combine with above-mentioned purification and enriching step.Can carry out the isoelectric focusing of sample with modifying device shown in Figure 14.Improved place is included in the isoelectric focusing bar 610 that enriched layer 50 uses more than 1.The isoelectric focusing bar can be placed on first direction 620 of device continuously.Discrete isoelectric focusing bar more than two can be placed on the second direction vertical with first direction 630.Will be with two or the above parallel placement of bar.With each isoelectric focusing bar 610 be placed on compression layer 510 below, ceasma 660 is arranged below the center of enrichment ceasma in compression layer 510.Dotted line 640 among Figure 14 has provided the central shaft of each enrichment bar 610.Each ceasma 660 can be regarded the elongation of hole 518 in first direction 620 as.The central shaft of each enrichment bar 640 is directly below the ceasma 660 of impermeable compression layer 600.In the place that two above isoelectric focusing bars 610 are arranged, the ceasma 660 more than two is arranged, each ceasma is arranged along the central shaft 640 of isoelectric focusing bar 610, and is parallel with mutual ceasma in fact.

Can on any suitable static matrix, carry out isoelectric focusing (IEF), such as post bed, agarose or the polyacrylamide gel of cellulose (or glass) or other carbohydrate tunica fibrosa, sephadex particle.Static phase can prevent fluid convection, and stable p H is provided gradient, and the technology of this isoelectric focusing is known.For example: the analysis and the preparation of P.Glukhovskiy and G.Vigh. isoelectric focusing enantiomer separation.Analytical chemistry 1999,71, (17), 3814-3820.

In brief, IEF forms the pH gradient in the water electrolyte, and the segment distance of being separated by between two electrodes is done anode another is carried out electrophoresis as negative electrode for one.Can add acid medium (as high pH) at anode tap, add more alkaline medium (as low pH) at cathode terminal and form the pH gradient.Also can allow electric current pass through electrolyte naturally, form the pH gradient to another electrode from an electrode.This is that water is oxidized at anode because according to equation (4), and pH reduces naturally:

(4)

On the contrary, according to equation (5), water is reduced at negative electrode, and pH raises:

(5)

Can make the pH gradient obtain stable and linearisation by the method that in electrolyte, adds ampholytes.Ampholytes is the molecule of both sexes, have can be replaced acidity and basic group.In order in the IEF process, to set up stable p H gradient, can use the mixture of different ampholytes.Every kind of ampholytes has different isoelectric point (pI), and it is the pH when net charge equals zero on the ampholytes.

Ampholytes moves in extra electric field, arrives the pH gradient place of their pI up to their.Their speed becomes zero then.The pH that they can cushion any outside changes, for example when the analyte in the sample enters gradient.When applying positive voltage, be arranged the most close anode of material of minimum pls, the most close negative electrode of material of the highest pls in the electric field of different ampholytes between anode and negative electrode in a continuous manner with respect to the negative electrode anode.The pH of Fang Li be the ampholytes mixture of 3.0-10 from sigma chemical company, for example number is the product of p1647.

Available covalent bond is fixed to ampholytes on the matrix, forms Stationary pH gradient (IPG) on bar or flat board.

For example, can buy such IPG strips bar from Amersham Biological Science Co., Ltd.The Amersham number is the product of 17-6003-73 can be used to form pH3-11 on 7 centimetres distance a gradient.Remove the IPG technology and have that electric current is weak, stability is high, ampholytes does not flow out outside the characteristics of the not analysis of interfere with subsequent together with analyte, it is similar that IPG technology and ampholytes move formation pH gradient.

For electrically charged analyte is carried out isoelectric focusing, analyte is added in the pH gradient of isoelectric focusing bar 610, and the adding ampholytes uses the same method.The same with ampholytes, analyte analyses that thing is moved to their isoelectric point and by enrichment.Under stable state, charged enrichment of analytes arrives at isoelectric focusing band 460.At this point, isoelectric focusing stops.

Then porous layer 60,70 and 80 be assembled into compression layer 600 below, added top electrodes (140) and bottom electrode 500, add the water buffer solution of q.s, make ion pass porous layer and contact with electrode.Apply suitable bias voltage (bias potential) as previously mentioned, enter the arrowband of catching layer 60 by the crack 660 of passing compression layer, analyte is by further enrichment in the isoelectric focusing band.After analyte is captured to capture film, relieving attachment 2, capture film 60 as previously described, uses suitable MALDI matrix solution and dries to make it to form the MALDI crystallization with analyte as skateboard component, is put into then on the MALDI sample panel.

According to said method, analyte is enriched in first direction 620 by isoelectric focusing in electric field, and then vertically 650 electric field is enriched to discrete molecules line predetermined on the capture film, and the definition of molecular line is corresponding crack 660 in the compression layer 600.Therefore can under high detection sensitivity, detect the mass spectrum property of analyte easily.

Embodiment 1

Separation is carried out the MALDI-TOF analysis from sero-abluminous ubiquitin polypeptide on the PVDF film, improve the mobility of polypeptide with lauryl sodium sulfate (SDS)

The protein standard of mark:

With red (the Cat No.T-6134 of Texas, Molecular Probes, Eugene OR) NHS-ester and Marina Blue (product# M-10165, Molecular Probes, Eugene OR) prepares the ubiquitin (TR-ubiquitin) and the Marina Blue bovine serum albumin(BSA) (MB-BSA) of Texas's red marker respectively.The mark program of using Molecular Probes to recommend.In brief, about 1 g/ml inserts in the dimethyl formamide (DMF) with the NHS-fluorescent marker, the concentration that joins in the neutral standard phosphate buffered saline (PBS) by stirring is in the protein example of lmg/ milliliter or higher concentration then, and the mol ratio that makes labelled reagent and protein is 10/1.Make label and protein example reaction one hour.Then reactant mixture by through the centrifugal post of P6 of 250mM L-histidine (sigma) buffer solution balance (spin column, Bio-Rad).With the protein example 25 microlitre equal portions packing of mark, chilled storage.

Electrolytic cell, polyacrylamide, agarose and perforated membrane:

With light electroblotting method (photo-electroblotting) separate and the fundamental system of enrichment of analyte as shown in Figure 6.Open-ended sample cell is that the polystyrene tubule by the short cylinder of about 2 centimeter length and about 2 cm diameters forms.Separating layer forms the bottom of sample cell attached to an end of pipe.Separating layer is made by the 2-3 millimeter sheet (obtaining from biorad) of 10% polyacrylamide precast.Polyacrylamide sieves as molecular weight, and the electrophoretic velocity that postpones large protein surpasses small protein and polypeptide.Can reduce the crosslinked of polyacrylamide, it is crosslinked to postpone the migration velocity of albumen and polypeptide better to allow large protein migration quickening maybe can improve, and the technology of electrophoretic separation protein is well-known in this gel-type vehicle.

Randomly, before sample adds, with the edge of 1% agarose sealing polyacrylamide gel and the sidewall (in order to prevent leakage) of sample cell.Before using, agarose and polyacrylamide 250mM histidine buffering liquid (pH 7.8) balance.Perhaps, acrylamide gel can in-situ polymerization, does not need encapsulant.Agarose (Type 1-B:low EEO) is from Sigma Chimical company.Usually, the agarose that weighs up 100 milligrams is put into the vial of being with screw-cap.In order to promote the electromobility of protein and polypeptide, add lauryl sodium sulfate (SDS) during use, make their the less influence that is subjected to surrounding environment pH of electromobility.When using SDS, the L-histidine Sigma that will contain 10% lauryl sodium sulfate aqueous solution (SDS), 10 milliliters of 250mM (Sigma) of 10 L is added in 100 milligrams the agarose.(if when not using SDS, then not adding SDS).Before using, agarose is heated into liquid state in microwave oven.Sidewall along the polystyrene cylinder is poured on one deck agarose above the prefabricated polyacrylamide, to seal all leakages.Cooling off agarose then makes and solidifies.Then, sample is added in the L-histidine that contains 10% glycerine 250mM of 1-10 microlitre.Then one deck running buffer (identical with agarose solution above-mentioned, as still not contain agarose) carefully is added to above the sample.

For the separation of carrying out analyte molecule with catch, with prefabricated polyacrylamide be put into a series of perforated membranes 8 above.Film is as described below, buys and stores, and is that do or pre-hydrated.Film is cut into square (1.5 centimetres of about 1.5 cm x), uses methanol rinse, with moistening and clean hydrophobic polymer.After in transferring to aqueous buffer solution (250mM histidine), their are refrigerated until use.PVDF dialysis membrane (molecular cut off 250,000), regenerated cellulose dialysis membrane (molecular cut off 1000) and CEA dialysis membrane (asymmetrical fibre plain ester) molecular cut off 500 be all from Spectrum Laboratories, Rancho Domingez, CA.PDVF-P (0.45 micron pond footpath) and PDVF-PSQ (0.2 micron pond directly) be from Millipore, Billerica, Massachusetts.Zeta film (0.45 micron pond footpath) is from BioRad, Herc μ les, CA.

Semiconductor and optical instrument

N type germanium wafer (thick 14mil, 5 centimetres of diameters) is available from the Polishing Corporation company of the U.S..The resistivity of wafer is less than 0.4 ohm-cm.Upper surface at wafer uses gallium/indium eutectic and copper wire to make ohmic contact.。Ohmic contact was with fast dried epoxy adhesive layer mechanical consolidation in five minutes.As the reinforcement germanium wafer of light anode, be installed on the optical table with laser and focusing optics together.The germanium wafer of installing is connected on the support of laboratory with clip.He-Ne light with 633 nanometers of 1 milliwatt power carries out the photoelectricity trace.With 100 microns surfaces of impacting bottom surface glass to the convection light between the 1mm of diameter.

In this embodiment, the germanium wafer of reinforcement is used as photoresponse electrode 100, shown in Figure 100.Resilient coating 80 is by being formed by the saturated PVDF-P film of 250mM histidine cushioning liquid.On the PVDF-P film, place the film formed barrier layer 70 of dialysing by CEA.Catching layer 60 is made of aforesaid PVDF-dialysis membrane.Polyacrylamide separating layer 40 forms sample cell with the above additional materials, directly is assembled into and catches on layer, resilient coating and the photoresponse electrode.

Electrophoretic separation and hybridization

The TR-ubiquitin of 0.2 μ L, MB-BSA (1-2 μ g/ μ L) or both are added in the 2 μ L samples, and sample contains the 250mM histidine aqueous buffer solution that comprises 25% (v/v) glycerine.The mixing sample was approximately rotating 2 minutes under the 1000xg.The supernatant of about 0.5 μ L is used for separating and electricity hybridization.The result of the mass spectrum result of protein mixture and the standard protein of purifying or polypeptide sample (only comprising ubiquitin and 1%TFA) is suitable.The ubiquitin sample of purifying (standard) directly is diluted among the 0.1%TFA (reaching 800fmol/ μ l or 10fmol/ μ l),, after solvent evaporation, does not have interfering material to exist so that before carrying out the MALDI mass spectral analysis.

In order to carry out electrophoresis, the ohmic contact on the photoresponse electrode is connected with pressurizer (Princeton application study).Between sample and top light response electrode, placed the film combination that is in " moistening/buffering " state.On rete, put a plastic cylinder (diameter is 2 centimetres, highly is 2 centimetres).The agarose that adds heat makes liquid reach the height of 3-5 millimeter.In case agarose solidifies, and adds the high water buffer solution of another 3-5 millimeter (0.01%SDS of 250mM histidine).The counterelectrode (platinum, palladium or carbon) at electrolytic cell top is immersed, keep the approaching of itself and agarose.Add the sample (TR-Ub or MB-BSA) contain 25% glycerine and 75% histidine buffering liquid, form with the circular counterelectrode at electrolytic cell top and electrically contact.Remaining separates and the enrichment program is to carry out in the darkroom.

Between photoresponse electrode and counterelectrode, apply bias voltage, light source (4 milliwatt diode lasers emission 600-700 nano wave length) focuses on the point of about 50 micron diameters of the back side (low) formation of wafer, the counterelectrode central vertical at its center and electrolytic cell top.Usually the inclined to one side electricity that imposes on germanium electrode (with respect to the counterelectrode of platinum) is+4 volts, can produce the total current (wherein 40% is photoelectric current, and the electric current when dark approximately is 300 μ A) of about 500 μ A.Usually the process that allows electrophoretic separation and catch was carried out about 9-20 minute.

After separating and enrichment finishes, shift out counterelectrode and top buffer solution in the sample cell.Remove the acrylamide separating layer then, capture film was immersed in deionized water (or preferably immersing 0.1% trifluoroacetic acid) middle 2-5 minute, remove salt.Then capture film (as the PVDF film) is sticked on the stainless steel MALDI sample panel with the 3M two-sided tape.Then matrix solution is added on the film, air-dry.Sample panel is put in the mass spectrometer analyzes.

Stainless steel MALDI sample panel is directly carried out MALDI-MS analyze, the matrix solution that comprises 0.5 to 2.0 μ l of sample directly is added on the stainless steel MALDI sample panel, and is air-dry.Then sample panel is put in the mass spectrometer and analyzes.

The analytical method of MALDI-MS

The QGEN_PR2 method that use provides is analyzed in ABI/Perceptive Biosystems VoyagerDE (MALDI-TOF) instrument.Use CHCA to be matrix solution, imposing a condition is the 25kV accelerating voltage, and 89% gate voltage (grid voltage), 0.25% wire voltage, 200ns postpone, 2800 laser, 64 on average scans, 3.45e-7 holder, 100 low quality grids, anion are deducted.

The result

As mentioned above TR-ubiquitin direct sunshine-electricity is enriched on the CEA dialysis membrane and (does not have pvdf membrane).Mass spectrogram shown in Figure 16 is that the oligopeptides that will extract from film is added to the MALDI matrix solution that contains CHCA, and solution directly is added to air-dry back acquisition on the corrosion resistant plate.Multimodal has been represented covalent bond each red ubiquitin polypeptide of Texas of 0,1,2,3 or 4 molecule.

Be used for comparison, Figure 17 has shown the mass spectrogram of identical ubiquitin sample, this sample is through light-be enriched to and hold back 250, (support (backed)) on the PVDF-dialysis membrane of 000MW by the CEA dialysis membrane of holding back 500MW, after the photoelectricity hybridization, water cleans the PVDF dialysis membrane, drying, and the MALDI matrix solution that will contain CHCA then directly is added on the example enrichment site.Matrix solution be evaporated air-dry after, directly use (3m) two-sided tape to paste on the stainless steel MALDI sample panel PVDF dialysis membrane, be put into and carry out MALDI-MS in the mass spectrometer and analyze.Figure 16 and 17 has shown two main distinctions.At first, when with the PVDF film, the intensity of the ion flow at maximum intensity peak is maximum.This shows that capture rate is maximum if during with the PVDF film.The second, catch with two kinds of different films, can see mark the ratio of the red ubiquitin of the Texas of 0,1,2,3 or 4 molecules be different.Catching what obtain with the CEA film is less molecular weight with every mole of ubiquitin of Texas's red marker of 0 or 1 molecule.Therefore show the CEA dialysis membrane more preferably in conjunction with Texas red-the ubiquitin molecule of protrude mark (how negative electric charge).On the contrary, by with the comparative result analysis (not video data) that directly the ubiquitin sample of mark is added on the stainless steel MALDI Target Board, show the PVDF film it seems more preferably in conjunction with underlined ubiquitin molecule.

Embodiment 2

Serum polypeptide with the suitable film separating belt negative electrical charge from human serum of MALDI

A subject matter of low abundance polypeptide is the existence that high-abundance proteins has been covered low abundance polypeptide in analyzing blood, blood plasma and the serum.Existing report is removed the albuminous while of high abundance and has also been removed a lot of low abundance polypeptide from the sample of blood, blood plasma and serum.Therefore the method that finds a low abundance polypeptide to separate with albumin is very important task.In order to promote to dissociate, use the detergent treatment blood serum sample in this research.

The human serum sample

The blood serum sample of preparation detergent treatment: in Eppendorf microtubule (500 μ L volume), 5mg/mL octyl group-β-D-glucopyranoside (OG) is added in the 10 μ L human serums (from Sigma ChemicalCo.).Then, the serum of detergent treatment 4 ℃ of overnight storage, is placed on room temperature after 14 hours.(in the 250mM histidine buffering liquid as tracer) and 0.5 μ L glycerine prepare sample to use serum preparation sample, 1 μ L 250mM histidine buffering liquid, the 1 μ L Texas of the detergent treatment of 3 μ L red then-the Leu enkephalen of mark.Sample mixture under about 1000g centrifugal about 1 minute obtains the drop of 1 and 3 μ l.

Sample cell and separating layer

Then sample is put in the sample storage pond of Serum Profiler analytical cell so that separate, be enriched on the film that is fit to of MALDI, carried out mass spectral analysis.The storage pond of enricher is to be made of the sample loop that electron opaque material is made, and electron opaque material is as polystyrene, polyethylene, polypropylene, Merlon, polymethyl methacrylate, many methylpentenes, Teflon ^TMDeng, these materials form the sidewall in sample storage pond.

The bottom surface in sample storage pond is brought sample in the isolating construction of ion-conductance contact with it into, and this isolating construction is formed by one or more separating medium layers.In the present embodiment, separating layer is made by the 2-3 millimeter sheet (obtaining from BioRad) of 10% polyacrylamide precast.Polyacrylamide can delay the electrophoretic mobility of high molecular weight protein as the molecular weight sieve, but can improve the electrophoretic mobility of small molecular protein and polypeptide.Polyacrylamide can be than low crosslinking degree the electrophoretic mobility of high molecular weight protein to be accelerated, and also can be high crosslinked and delay the electrophoretic mobility of small molecular protein and polypeptide, and this is that the personnel of this area gel-type vehicle electrophoretic separation albumen are known.Randomly, the gel edge seals with 1% agarose.Agarose and polyacrylamide are used the 250mM histidine buffering liquid balance of pH 7.8 before use.(in addition acrylamide polymerization and do not need encapsulant in position.)

Catch layer, barrier layer and resilient coating:

Under the polyacrylamide separating layer, the ion-conductance contact is the layer of catching of catching the polypeptide analysis thing with it, and the polypeptide analysis thing comprises polypeptide, oligopeptides and protein.Catch is to be undertaken by the hydrophobicity effect of polypeptide and polymer capture film.By the film that porous Teflon or PVDF material are formed, catch very useful to the polypeptide that is undertaken by the hydrophobicity effect.In the present embodiment, capture film is formed (molecular cut off 250,000) by the PVDF-dialysis membrane, preserves down in 4 ℃ in the buffer solution aqueous solution (250mM histidine) with preceding.

Catch layer below with it the ion-conductance contact be the optional barrier layer that exists, it is used to prevent escaping of non-hydrophobic protein and polypeptide.CEA dialysis membrane (the plain ester of asymmetrical fibre, molecular cut off 500) as the barrier layer, is used preceding kept dry.Capture film and barrier film are cut into square (about 1.5cm * 1.5cm), with the preceding washed with methanol of using, clean with assurance.These two kinds of films are all from SpectrumLaboratories, Rancho Domingez, CA.Analyte is hunted down the back before the MALDI matrix solution that adds small size such as 0.3-2 μ l catching layer, does not need elution step.Therefore hydrophobic protein and polypeptide are caught by the PVDF dialysis membrane, and be irrelevant with the molecular cut off of capture film.

The buffer area:

Below catching layer and barrier layer, what contact with its ion-conductance is the buffer area, and the buffer area is to be used for cushioning the product that (or catching) electrode surface forms, and electrode is to be used for producing electric field to make analyte produce electrophoresis, separation and enrichment in capture film.Among the present invention, the buffer area is made of the Immobilon-P film (from Millipore Inc.) of an individual layer, and saturated with the 250mM L-histidine buffering liquid of pH 7.8.

The photoconduction electrode:

Contacting with the resilient coating ion-conductance and just in time being positioned under it is the photoresponse anode of being made by n-type germanium wafer.Available from U.S. Polishing Corporation company, resistivity is less than 0.4ohm-cm for germanium wafer (thick 14mil and diameter 5cm).Be installed in germanium wafer on the glass plate as work electrode and light anode with five minutes fast dried epoxy glues.Use gallium/indium eutectic to be connected with copper conductor with the ohmic contact that copper cash is made on wafer.Ohmic contact is reinforced with five minutes fast dried epoxy adhesive layer mechanicalnesses.

The method that the human serum sample analyzes:

For separation and the combination of carrying out sample, in the sample cell on acrylamide/agarose layer top, add the sample mixture of a pipe (0.5-8 μ l) through detergent treatment.Directly put an annular then into the loose counterelectrode that contacts of pond inwall (negative electrode), this counterelectrode (negative electrode) is made of platinum.Platinum cathode and photoresponse anode and pressurizer (Princeton Appllied Research, model 273) connect, and in the darkroom (room darkened), general+4V bias voltage is used for photoresponse anode and platinum cathode.Helium/neon 633nm light with the output of 1 milliwatt carries out the optical excitation of photoresponse anode (rayed is on the relative sheet of electrolyte) backside surface.Made laser focusing system, strengthened the laser of irradiation glass bottom, the diameter of this laser focusing light beam arrives in the 1mm scope at 100 microns.The bias voltage of 4V and irradiation have simultaneously produced the total current (about 30% is photoelectric current) of 700mA.Before the bias voltage zeroing, carry out about 20 minutes separation, separately connected the lead of upper/lower electrode then.

Then, take collection chamber apart, detect the fluorescence of glue and film.If acquisition procedure is thorough, all fluorescence are included in the capture film.Epipolic point is with in cutting-out, immerses in the deionized water several minutes.Pvdf membrane is air-dry, carries out maldi analysis by the method that examples of implementation 1 are described.Press the method that examples of implementation 1 are described, directly in Voyager DE work station, the protein that derives from pvdf membrane of catching is analyzed.

Figure 18, shown in the 18a and 19 is with CHCA or the protein of sinapic acid analytic band negative electrical charge and the normally result of polypeptide in the MALDI matrix solution.With the CHCA in the MALDI matrix solution 1000 to 15,000 daltonian low-molecular-weight peptides and polypeptide are carried out mass spectral analysis and can obtain best result.On the contrary, the sinapic acid in the matrix solution is improved the signal of HMW (＞15,000 dalton) molecule.Therefore, each in analyte and this two kinds of stroma ground substances carried out maldi analysis respectively, can all provide ideal results for low-molecular-weight and high molecular weight molecules.

Embodiment 3

On the PVDF film, the positively charged polypeptide from the human whole serum is carried out electrophoretic separation to carry out

MALDI-TOF analyzes

Similar with embodiment 2 to embodiment 1, in the present embodiment, polypeptide among the human serum sample and micromolecule serum proteins are enriched on the capture film by electrophoresis by gel, thereby macro-molecular protein is separated with small protein matter with the micromolecule target polypeptides.The same embodiment, charged polypeptide and protein molecule move in the electric field that adds anode and negative electrode.In the present embodiment, positively charged analyte molecule is to electronegative cathodic migration, obtains enrichment and combination with it at the trapping region 68 of capture film 60.The same embodiment is described, is directly analyzed from the positively charged protein of being caught of capture film.The same embodiment 2 is described, carries out maldi analysis with Voyager DE mass spectrometer.

The main distinction of embodiment 1,2 and present embodiment 3 is that the photoconduction electrode is optional in present embodiment 3.Substitute as a kind of, similar to foregoing top electrodes 140, non-photoconduction electrode substance also can be used as bottom electrode 500, and bottom electrode can be used as negative electrode or anode.Though the photoconduction electrode is optional in certain embodiments, need to have the compression layer 510 in hole 518 at least, it is used for the trapping region 68 of enrichment of analytes to capture film 60.As shown in figure 13, preferably, also can use second compression layer 520.Compression layer is impermeable for ion flow, can only make target analytes be enriched to the capture region 68 analyte upper storage reservoirs of capture film 60 by hole 518.Therefore, do not need the guide effect of photoconduction electrode.

Material:

Bottom electrode (500): cover palladium (Pd) thin slice with Kapton (polyimides) adhesive tape, in bottom electrode, reserve the conductive area that needs.For embodiment, the hole of the 2mm diameter that leaves in the Kapton adhesive tape is used for exposing the palladium of this position, as the surface of bottom electrode.

Top electrodes (140): top electrodes is made of platinum (Pt) line, is the toroidal of 3mm diameter.In order to mate, top electrodes is connected with the control electrode of the pressurizer or the electrical equipment that becomes and the lead of reference electrode with the pressurizer of 3 utmost points or the electrical equipment that becomes.

1 " * 3 " the Kapton adhesive tape on bore 2.4mm the hole prepare bottom electrode (500).Adhesive tape is placed on fritter palladium (Pd) thin slice, and both are put on the microscopical glass slide then.The work electrode lead of pressurizer is directly linked thin slice by clip.

Resilient coating (80): resilient coating is made up of the Immobilon-P film, from Millipore, and Inc.The Immobilon-P film carries out saturated with the 250mM L-histidine aqueous solution of pH 7.8.

Barrier layer (70): molecular cut off 500, the CEA dialysis membrane is from Spectrum Laboratories, Inc..

Catch layer (60): molecular cut off 500, the dialysis membrane of polyvinylidene fluoride (PVDF-DM) is from Spectrum Laboratories, Inc..

Separating layer (40): 10% polyacrylamide, from Bio-Rad Laboratories, Inc..

The gel of being made up of the low EEO agarose of 1% in saturated DL-glutamic acid and the DL-asparagine aqueous acid (from Sigma Chem.Co.) is used for the sidewall (12) of hermetic separation layer (40) and sample cell (6).

Sample buffer: use the aqueous buffer solution dilute sample, top electrodes and bottom electrode are electrically contacted.With the L-glutamic acid and the saturated buffer solution aqueous solution of DL-asparatate (from Sigma Chem.Co.), the pH of buffer solution is about 3.0.

Sample cell (6): polycarbonate cylinder is used to make the separation and the enricher in single pond at the bottom of the band of upper opening.Single pond is 19.15mm O.D. * 16.95mm I.D. * 17.32mm height.

Sample: 1 μ l glycerine+1 μ l ubiquitin (from Sigma Chem.Co.), its with reference to the explanation of manufacturer with red mark (Molecular Probes, Inc.)+8 μ l human serum (from Sigma Chem.Co.)+≈ 1 μ g octyl group-β-D-glucopyranoside (from Sigma Chem.Co.)+3 μ l sample buffer of carrying out of Texas.In case add all the components of sample, centrifugal and vortex mixing (mixing).The sample of 5 μ l is added to sample cell by pipette then.

The MALDI matrix solution: a MALDI matrix solution is a-cyano group-4-hydroxycinnamic acid (CHCA) saturated in 50% acetonitrile and 0.1% trifluoroacetic acid.The 2nd MALDI matrix solution is the 10mg/mL sinapic acid (SA) that is dissolved in 0.1% trifluoroacetic acid of 50% acetonitrile and 50%.The all material of MALDI matrix solution is from Sigma Chem.Co..

Instrument:

Electrode bias or Current Control: use from EG﹠amp; The 273 model pressurizers of the G Princeton Applied Research/electrical equipment that becomes carries out the operation of the voltage stabilizing or the power mode that becomes.

The acquisition of data: from the Powerlab/4SP of Dell, AD instrument and desktop computer.

The MALDI mass spectrometer: Voyager DE Biospectrometry Workstation, (AppliedBoosters, Inc.).

The preparation of film:

In sample separation, enrichment be attached to PVDF capture film (60) before, pvdf membrane soaked in methyl alcohol 1 minute.Secondly because pvdf membrane is a hydrophobicity, the effect of methyl alcohol is at first to make film wetting, can also be used for cleaning film.Then moistening pvdf membrane is placed in the deionized water and preserves.Preparing gel separating layer (60) with prefabricated 10% polyacrylamide from Bio-RadLaboratories.Take out polyacrylamide from box, the polycarbonate cylinder that is used as sample cell sidewall (12) is cut into discoid pond.The disk that downcuts was soaked liquid with the CEA film as barrier layer (70) in deionized water.The high salt concentration of prefabricated polyacrylamide gel need soak liquid in water, make conductance reduce to suitable scope.Though device can be operated in the conductance scope of very wide separating layer and sample buffer, such as 1micro-siemen to 100milli-siemens, but the device the ideal operation scope be 10micro-siemens to 10milli-siemens than close limit.

The structure of device:

In the following order porous layer is placed on to build on the bottom electrode and separates and enricher (2): sidewall, water buffer solution and the top electrodes of the agarose gel hermetic separation layer of resilient coating, capture film, compression layer, separating layer, heating.The sidewall that the agarose of heating is added to around the sample cell reaches the 1-2mm degree of depth.When agarose gel solidifies, will carefully be reduced in the pond as the platinum annulus of top electrodes, make its contact agarose.The top electrodes that adds enough water buffer solution covering platinum annulus then.This schematic representation of apparatus sees 6.

The operation of device:

(3-D-glucopyranoside+3 μ l water buffer solution, preparation comprises the sample of human serum to add ubiquitin+8 μ L human serums+＜1 μ goctyt-of 1 μ l glycerine+1 μ L Texas red marker.Solution is behind simply centrifugal, vortex, and 5 μ l samples are added in the cell by the center of platinum annulus.The bias voltage of bottom electrode is-5V.The continuous monitoring electric current.After the beginning transient state, in follow-up 45 minutes separation and enriching step, current stabilization is to about 200 μ A.The ubiquitin of the Texas's red marker analyte that serves as a mark is followed the tracks of the process that analyte separates and catches.Regulate the time and the electric current that are fit to separation, to reach optimal separation and to catch the result.

When separating step and enriching step end, with bias voltage set to zero, relieving attachment.Use Mineralight, the 366nm projected light of the fluorescent lamp of Entela UVGL-58 is observed fluorescently-labeled tracer protein, as the ubiquitin of the Texas's red marker on the capture film.Downcut the phosphor dot on the PVDF-DM capture film, immersed deionized water 2 minutes, dry under nitrogen.In order to paste on the MALDI sample panel, film is divided into the small pieces of 2 or a plurality of 2 mm square.When small pieces are put on the stainless steel MALDI sample panel, have the CHCA matrix that has added 1 μ l on 1 small pieces at least, add the SA matrix of 1 μ l on remaining small pieces.In case matrix solvent bone dry is put into film in advance with clean tweezers and sticks on the 3M two-sided tape of 2 mm square on another stainless steel MALDI Target Board with the precalculated position.

The MALDI-mass spectral analysis:

The mass spectrometric parameter of Voyager DE is as follows: 20, and the 000V accelerating voltage, 94.1% gate voltage, 0.05% wire voltage, 11ns postpone, source, laser apparatus is decided to be 2800, on average number of scans 64, anion are deducted.

The result:

Figure 20,20a and 21 have provided the MALDI mass spectrum result of typical positively charged polypeptide and protein.As seen from the figure, detected ubiquitin tracer protein (referring to 8600,9330 and 10, the 100 mass spectrum units) signal that comprises mark mass spectra peak in interior a large amount of low molecular weight protein (LMWP).Figure 18 has shown with sinapic acid as MALDI matrix (the easier moderate protein molecule ionization that makes greater than 20,000 molecular weight) to have only moderate albumin (about 68,00 mass spectrum units) to occur.

The little mass spectra peak at visible in addition 34,000 mass spectrum unit places.Except that these two mass spectra peaks, visible several greater than 11,000 daltonian mass spectra peaks, come from the macro-molecular protein that separates with micromolecule polypeptide in the polyacrylamide gel separating layer beyond doubt.

Embodiment 4 catches porous in the array of analyte attached to electric enrichment hole of capture medium

In a specific embodiment of the present invention, the hole of the two-dimensional array on the solid material is to be used for charged analyte to the hole of electrophoresis enrichment to locate.With the porous media of catching analyte attached to the place, hole (outlet or porch or within) to catch the analyte of enrichment.Each hole is surrounded into the presumptive area of catching medium by the atresia circumference.The atresia circumference is used for keeping solution and avoids taking bigger zone in presumptive area.Device with one or more holes in use just has following steps:

1.) deposition step, charged analyte deposits with the capacity of keeping sample as liquid, and it is placed in the contacted electrolyte of negative electrode with the anode of Kong Yiduan and the hole other end.

2.) enrichment and catch step, analyte is driven and enrichment in the hole by electrophoresis.Still charged analyte is captured to and places the porous of Kong Chu (in the hole or at the outlet or the inlet in hole) to catch on the presumptive area of medium in advance in this step, thus the charged analyte of enrichment in presumptive area.

3.) analytical procedure is to being analyzed by the charged analyte of catching of enrichment in presumptive area of obtaining in the step in front.

Analytical procedure also comprises following inferior step:

A) catch in the medium to porous and add the fluid analysis matrix solution that comprises liquid flux and dissolved matrix material, make analyte be discharged into the upper surface of liquid flux and porous media,

B) porous is caught in the presumptive area of medium, with atresia circumference liquid hold-up solvent,

C) evaporating solvent is stayed in the presumptive area of porous media upper surface the stroma ground substance of dissolving and the analyte of catching,

D) feature of the analyte of analysis presumptive area upper surface.

Usually analytical procedure comprises further that also hole and accompanying porous are caught medium to be inserted in the mass spectrometer of analysis of analytes.And mass spectrometer normally has analyte and matrix is carried out the auxiliary MALDI mass spectrometer of the laser of desorption altogether of laser.Analyte is protein normally, and polypeptide is or/and peptide.The hole is normally with the substantially the same hole of array format.

The electrophoretic apparatus that describes below can be united use with the hole array, carries out step of the present invention.What further describe is to can be used for making up and using the material of hole array and the embodiment hole of method.

The following examples A) the porous capture film in is (from Millipore Corporation.Billricia with porous polyvinylidene fluoride (PVDF), MA) as the Immobilon-PSQ film, the hole array is (from McMaster Carr Corporation at solid phase PVDF, Atlanta, thin slice GA) is made.As described, perforated membrane is welded to preparation atresia circumference on the solid phase thin slice all around in the hole with thermal weld.

The Immobilon-PSQ film is welded on polyvinylidene fluoride (PVDF) sheet:

In the present embodiment, having described will be such as the several different methods of porous PVDF capture film type of permanent attachment on solid phase PVDF sheet (film) of Immobilon-PSQ.These methods comprise perforated membrane are thermally welded to such as by 0.010 " on the solid material of the polymer sheet that thick solid phase PVDF makes.In the method, perforated membrane is placed on the array of orifices on the solid polymer sheet.The function in hole is the material that makes absorption, such as polypeptide and protein, utilizes solvent streams to arrive the upper surface of porous capture film through the hole.

Can weld by the heating die arrangement that Figure 27 provides.Desirable imprinting apparatus needs temperature control equipment, in individual special temperature range inner control temperature, has at least a film soldered when surpassing melting temperature and being lower than autogenous ignition temperature.In addition, the welding impression generally has flat territory, base area to exert pressure to the surface of at least one film.But generally at the impression center, duck eye is arranged also, be used for the formation of part all around welding girth in the not welding of film.Impressing conventional flatiron available as that describe below makes.Being preparation welding impression, gets into the cave at head then in the bottom of removing conical flatiron tip, and the long 18G hypodermic injection of 5mm is just in time passed this hole with syringe needle.Solder horn is placed in the drilling machine then, bores the inside of pond polishing hypodermic injection with syringe needle with 62 ° of high speed stainless steels.

When welding, pay special attention to make the hole in the soldering appliance aligning PVDF sheet.Can prepare drill jig (jig) and drill bit is aimed on the array in 25 holes, on identical duck eye, film be carried out suitable thermal weld with Welding iron then, as shown in figure 28.Drill jig is made up of 3 parts, the polycarbonate substrates flat board, aluminium drill bit flat board (array that 25 duck eyes are arranged, 0.024 " diameter) and aluminium Welding iron flat board (array that 25 duck eyes are arranged, 0.052 " diameter).

The PVDF perforated membrane is welded to the method for PVDF immobilon-p:

" thick solid phase PVDF sheet pastes the center of polycarbonate substrates flat board with 45mm * 45mm, 0.01 with the adhered layer of 3M two-sided tape.

Aluminium drill bit flat board is screwed on the substrate on the PVDF sheet.

With design in the drilling machine use 0.024 " drill bit of inch diameter and needle pliering are by boring the array that solid phase PVDF sheet forms 25 holes.

Removal drill bit flat board, the Immobilon-PSQ film is placed on the hole, and the Welding iron flat board is screwed on the substrate.Hole in the Welding iron flat board is alignment on the hole in the PVDF sheet just in time.

Flatiron is transferred to about 850 , is incubated several minutes.

Firmly solder horn is advanced in the hole of aluminium sheet middle plateform, up to touching film.Hold flatiron 1-10 second facing to film up to producing acceptable welding.

After finishing welding, remove flat board, use the microscopic examination film the film on all 25 holes.

Use tweezers, remove pad film on every side.

Then the array that welds is dipped in and carries out sonication in 10 minutes in the methyl alcohol.

After having the sheet that welds film air-dry, put into polybag in order to using.

Figure 29 and 30 has provided the film welding and has contained the application result of the solution of MALDI stroma ground substance.

The mass spectral analysis of the sample that extracts: the PDVF-PSQ film is welded to hole on the solid phase PVDF sheet with said method.With the wetted with methanol of perforated membrane with 1 μ l, the analyte that will contain the polypeptide fragments of ACTH is put into the front (one side relative with solid phase PVDF) of the perforated membrane in about 0.5 μ l PBS aqueous buffer solution then.Analyte is with containing 80 of sinapic acid (SA) matrix: 2 * 0.3 μ l MALDI matrix solutions that the mixture of 20ACN/ water (being dissolved in 80% acetonitrile with 10mg/mL, in 20%0.1% trifluoroacetic acid) is formed arrive the apparent surface with analyte stream.All MALDI matrix solutions are from Sigma Chem.Co..MALDI-TOF is carried out at the back side of film to be analyzed.The results are shown in the mass spectrogram of Figure 31.

Embodiment 5

In the hole of solid phase film in the porous integral castable plastic enrichment and the protein analyte of catching and

Be used for the subsequent extracted that MALDI-TOF analyzes

Figure 32 has provided the basic structure schematic diagram with subsequent extracted of catching of the protein that is used for the MALDI-mass spectral analysis and polypeptide analysis thing.This structure is made up of the plastic frame that comprises one or more micropores.Hole or perforation are preferably minor diameter, are generally 1 micron to 2 millimeters.Penetration hole diameter more generally is between 100 microns to 1 millimeter.The lower part of cell is coated with porous protein capture film, as PVDF-PSQ (the Immobilon film, from Millipore Corp., Billericia, MA).With one or more encapsulating methods capture film is sealed on the plastic frame.The simplest seal is made up of locating ring, as the nylon washer suitable mutually with locating ring.After adding sample and solvent, cover cell with cover glass.

Method: the ubiquitin (in water) of (about 10 picomoles) Texas red marker is added to the top of dry PVDF-PSQ film on a small quantity.Sample is dry under vacuum.Add 40 μ l acetonitrile/water solution, cover lid on plastic frame to sample.From following outflow solution and evaporation.This step repeats 2 times, so each sample has all used complete soln.For the ease of studying, the optimum extraction effect of Tr-ubiquitin is studied with following solution.The acetonitrile that uses and the ratio of water (ACN/ water) are: 100: 0; 95: 5; 90: 10; 80: 20; 70: 30; 50: 50.

The result: fluoroscopic image comes from the top (point sample sample) of film and the lower part (sample of extraction) of film.Figure 33 A and 33B and 34A and 34B have provided some results.Solvent or optimum extraction effect are 80: 20.

Embodiment 6

Analyte is captured in the whole porous capture material that forms in the array of electric enrichment hole

In this embodiment, whole porous is caught material and is formed in the two-dimensional array of apertures in the solid matter.In this embodiment, charged analyte is enriched to by electrophoresis in the porous capture material of integral plastics of electric enrichment hole array.The porous media of catching analyte is cast in the duck eye of a series of diameter 200 μ m, this duck eye be in advance on polyvinylidene fluoride (PVDF) supporting layer boring and form.In case finish the porous polymeric effect, the madial wall in hole can be in order to support polymer material better.Porous mass carries out original position manufacturing with methyl methacrylate as polymeric materials.Usually, supporting layer be placed in Tefon-transparent under ultraviolet bag quilt quartz plate above, polymer material is added in the duck eye by sample injector.Quartz plate with Tefon-bag quilt transparent under another ultraviolet covers on it then, has avoided air bubble to exist.Form porous " filling " with one section appropriate time of this material of ultraviolet irradiation at last.Observe each point, find that porosity is consistent, the end face and the bottom surface of top and bottom supported layer flush (flush).

In our test, every whole porous is caught the site all through deionized water cleaning down and ultrasonic, removes residual polymer and other potential pollutant.After water cleaned, washed with methanol was all thoroughly used in each site, uses the 0.1%TFA rinsing then.Then, get the 500fmol ACTH segment 18-39 that is dissolved among the 500fmol BSA, catch the solution of getting that adds 0.5 μ l on the site to every whole porous with pipette.In case sample is air-dry on integral slab, water cleans each site, the saturated sinapic acid of 1 μ l of 80: 20 acetonitrile/0.1%TFA is added to the back side (the opposite one side that adds sample) of integral slab.At last, make that to catch the supporting layer of site array around whole porous thoroughly air-dry, be put in the MALDI mass spectrometer and analyze.Figure 35 has provided a single porous and has caught the site by the original position porous polymeric method original position drawing of rough casting.Figure 36 has provided several MALDI mass spectrograms of catching the site from 4 different integral porous in the array.In all examples, all detected target ACTH segment.

Claims (38)

1. enricher, it comprises:

End face, it comprises that at least one forms the hole in pond;

Catch layer; With

Separating layer, it is at end face and catch between the layer.

2. the enricher of claim 1, wherein catching layer is porous.

3. the enricher of claim 2, wherein to catch layer be film to porous.

4. the enricher of claim 3, wherein film is a hydrophobic film.

5. the enricher of claim 2 is wherein caught layer and is the dialysis membrane that the material by conjugated protein constitutes.

6. the enricher of claim 1, wherein end face comprises a plurality of holes, each hole forms a pond.

7. the enricher of claim 6, it comprises 10 to 100 ponds.

8. the enricher of claim 1, it comprises the porous layer between end face and separating layer.

9. the enricher of claim 1, it comprises at the porous layer of catching between layer and the separating layer.

10. the enricher of claim 1, it comprises a barrier layer contiguous with catching layer.

11. the enricher of claim 10, it comprises resilient coating, and is wherein catching between layer and the resilient coating on the barrier layer.

12. the enricher of claim 1, it comprises the porous layer between end face and separating layer.

13. the enricher of claim 1, it has the bottom surface with respect to end face, and wherein the bottom surface is an electrode.

14. the enricher of claim 13, wherein bottom electrode is the photoconduction electrode.

15. the enricher of claim 14, wherein the photoconduction electrode is single continuous semi-conducting electrode.

16. the enricher of claim 14, wherein the photoconduction electrode is discontinuous semi-conducting electrode.

17. the enricher of claim 1, it comprises that at the compression layer of catching between layer and the separating layer wherein compression layer comprises impermeability zone and at least one hole, and wherein said at least one hole is logical by porous material and hole, pond liquid liquid phase.

18. the enricher of claim 17, wherein at least one hole of compression layer is built-in with porous, suction, hydrophilic material.

19. the enricher of claim 17, wherein the impermeability of compression layer zone layer is adhered to mutually with catching.

20. the enricher of claim 19 wherein forms circular weld with the thermal weld method around at least one hole of compression layer, the impermeability zone that makes compression layer layer is adhered to mutually with catching.

21. the enricher of claim 1, wherein compression layer is positioned at the below of catching layer, and compression layer comprises impermeability zone and at least one hole, and wherein said at least one hole is logical by porous material and hole, pond liquid liquid phase.

22. the enricher of claim 21, wherein the impermeability of compression layer zone layer is adhered to mutually with catching.

23. the enricher of claim 22 wherein forms circular weld with the thermal weld method around at least one hole of compression layer, the impermeability zone that makes compression layer layer is adhered to mutually with catching.

24. the enricher of claim 1, it comprises first compression layer and second compression layer, wherein catches layer between first compression layer and second compression layer.

25. the enricher of claim 1, wherein first compression layer and second compression layer all contain a hole at least, and this at least one hole is logical by porous material and hole, described pond liquid liquid phase.

26. the enricher of claim 1, wherein capture film comprises at least a label that can be discerned by mass spectrometer.

27. the enricher of claim 1 is wherein caught layer and had the marker location that can be discerned by mass spectrometer, wherein the distance of precalculated position and marker location is known.

28. an enricher, it comprises:

End face, it comprises that at least one forms the hole in pond;

The bottom surface, it is the photoconduction electrode;

Porous is caught layer;

Separating layer, it is caught between the layer at end face and porous;

Compression layer, it is caught between the layer in separating layer and porous, and wherein compression layer comprises impermeable zone and at least one and at least one concentric hole, end face hole; With

Circular weld on the compression layer, wherein circular weld is at least round the hole of a compression layer.

29. the method with mass spectrometer determination and analysis thing comprises:

(a) provide enricher, this enricher comprises end face, and it comprises that at least one forms the hole in pond; Catch layer; With at end face with catch at least one separating layer between the layer;

(b) sample that will comprise a plurality of analytes is positioned in the enricher;

(c) form trapping region on the layer catching;

(d) trapping region a kind of analyte of enrichment at least;

(e) from enricher, take out separating layer;

(f) MALDI matrix is added on the trapping region;

(g) with on the analysis plates of enricher attached to the MALDI mass spectrometer; With

(h) with mass spectrometer the analyte of capture region is analyzed.

30. the method for claim 29 is wherein caught layer and had the marker location that can be discerned by mass spectrometer, the distance of trapping region and marker location is known.

31. the method for claim 29, wherein enricher comprises the electrode layer with photoconduction zone of aiming at cell, wherein in enricher, by inserting the counterelectrode that electrically communicates with sample, makes at least a analyte obtain enrichment at trapping region; Between electrode layer and counterelectrode, apply electric current; Make it make focused light source irradiation capture region by the guiding light source by the photoconduction zone.

32. the method for claim 29, wherein enricher comprises magnetic part, and MALDI spectrometer analysis plate comprises magnetic part, and wherein enricher links to each other by magnetic with this plate.

33. the method for claim 29 wherein navigates in the enricher sample by described sample is navigated at least one pond.

34. the method for claim 29, wherein, after at least one separating layer was taken out from enricher, drying was caught layer.

35. the method for claim 29 wherein only is applied to MADLI matrix on the capture region.

36. the porous capture film, it has the analyte of catching by focusing electric field on one or more predetermined discrete locations, and wherein the porous capture film is properly mounted on the MALDI mass spectrometer sample panel.

37. the film of claim 36, wherein the MADLI stroma ground substance is added on each discrete position, forms the crystalline solid of one or more MADLI matrix and analyte.

38. the device of the porous capture film of preparation claim 36 comprises:

(a) hold one or more cells of sample, sample is contacted with one deck separating layer at least thereby wherein contain electrolyte;

(b) porous capture film, it is positioned at the below of one deck separating layer at least;

(c) electrode layer has the photoconduction zone of aiming at cell;

(d) electrode electrically communicates with sample in cell;

(e) light source, its irradiation photoconduction zone causes on the predetermined discrete location of enrichment of analytes to the capture film in the sample.

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