CN1502116A - Compatible capture tandem mass spectrographic analysis equipment and method - Google Patents

Abstract

The invention provides an analytical instrument comprising an affinity capture probe interface, a laser desorption ionization source, and a tandem mass spectrometer. Also presented are new methods for protein discovery and identification and for characterization of molecular interactions that utilize the instrument of the present invention.

Description

Compatible capture tandem mass spectrographic analysis equipment and method

Technical field

The invention belongs to chemistry and biochemical analysis field, and be particularly related to equipment and the method for improving affine interactional evaluation and sign between analyte and analyte by tandem mass spectrum.

Background technology

With high-performance and the species analysis device electron spray ionisation (ESI) of coupling connection and the arrival of substance assistant laser desorpted/ionization (MALDI) technology mutually cheaply, allowed mass spectrum (MS) being used for the big molecular studies of biological correlation in the past ten years, comprise that protein purifies from complex biological system, standard analytical tools in occupy one seat.

For example, in a kind of technology that is called the peptide quality fingerprinting, mass spectrum is used to discern the protein of purifying from biological sample.Identification is to realize by the proteolysis fragment mass spectrum of protein purification is compared with the quality that goes out from the elementary sequence prediction that adds in advance the database.Roepstorff，The?Analyst?117：299-303(1992)；Pappin?et?al.，Curr.Biol.3(6)：327-332(1993)；Mann?et?al.，Biol.Mass?Spectrom.22：338-345(1993)；Yates?et?al.，Anal.Biochem.213：397-408(1993)；Henzel?et?al.，Proc.Natl.Acad.Sci.USA?90：5011-5015(1993)；James?et?al.，Biochem.Biophys.Res.Commun.195：58-64(1993)。

The similar data storehouse system of attack grows up, and it adopts (post-source) decay (PSD) obtains behind collision induced dissociation (CID) or the MALDI source fragment mass spectrum to discern the protein of purifying.Eng et al., J.Am.Soc.Mass.Spectrom.5:976-989 (1994); Griffin et al., Rapid Commun.Mass Spectrom.9:1546-1551 (1995); Yates et al., U.S.Patent Nos.5,538,897 and 6,017,693; Mannet al., Anal.Chem.66:4390-4399 (1994).

Permission also grows up to the mass-spectrometric technique of protein isolate to the small part rearrangement.Chait?et?al.，Science?262：89-92(1993)；Keough?et?al.，Proc.Natl.Acad.Sci.USA.96：7131-6(1999)；Reviewed?in?Bergman，EXS?8：133-44(200)。

Thereby being convenient to resolve proteomic image and digging is not subjected to the software resource of the sequence library of copyright restrictions to be easier to obtain to be beneficial to the evaluation of protein now on Internet.Protein prospector (http://prospector.ucsf/edu) is arranged among this, PROWL (http://prowl.rockefeller.edu) and Mascot search engine (Matrix ScienceLtd., London, UK, www.matrixscience.com).

Although high-precision mass distribution provides Useful Information---for example, adopt above-mentioned technology to help discerning the protein of purifying---this information still is restricted.A large amount of extra analysis ability are analyzed and the enzyme of target protein and/or the explanation that combines, carries out structural constituent of chemical modification, modification and the further protein identification after the translation by MS, will be released.

In addition, Fu Za biomaterial---for example blood, serum, lymph liquid, lymph, interstitial fluid, urine, juice, whole cell, cytolysis thing and emiocytosis product---the organic and inorganic salts that typically contain hundreds of biomolecule and hinder direct mass spectral analysis.Therefore, before observing, MS typically needs a large amount of sample preparations and purification step.

The standard method of sample purifying, for example liquid chromatogram (ion-exchange, size exclusion, affine and reverse-phase chromatography), film are separated, centrifugal, immunoprecipitation and electrophoresis, typically need a large amount of initial sample.Even when having obtained so the sample of volume, accessory constituent also trends towards losing in these purge processes, and it is to lose owing to analyte that non-specific bond and diluting effect suffer.These methods usually are high labour intensity.

Therefore, the needs of pair method and apparatus are arranged obviously, it is convenient to make the main and less important protein that is present in the heterogeneous sample all by Mass Spectrometer Method and need not in a large number liquid phase purifying in advance.Further also have the needs to the MS platform, it not only allowed the purifying of sample that is easy to get before mass spectral analysis, and allowed serial and parallel sample method of modifying.

These need partly satisfy by development affinity capture laser desorption ionisation method.Hutchens et al., Rapid Commun.Mass Spectrom.7:576-580 (1993); U.S.Patent Nos.5,719,060,5,894,063,6,020,208 and 6,027,942.This New Policy that is used for big molecule MS analysis has used novel laser desorption ionisation probe, and it has affinity reagent at least one surface.This affinity reagent is adsorbed desired analyte from heterogeneous sample, they are concentrated at detecting probe surface with a kind of form that is suitable for laser desorption ionisation subsequently.The purification process of off-line has been avoided in the coupling of analyte absorption and desorption, and permission is analyzed the initial sample of less amount and helped more at direct sample method of modifying on detecting probe surface before the mass spectral analysis.

Affinity capture laser desorption ionisation technology allows mass spectrometry applications in many standard biological quantitative analysis forms, comprises immunity, Nelson et al., Anal.Chem.67:1153-1158 (1995); And affinity chromatography, Brockman et al., Anal.Chem.67:4581-4585 (1995).Affinity capture laser desorption ionisation technology not only is applied to the research of peptide and protein, Hutchens etal., Rapid Commun.Mass Spectrom.7:576-580 (1993); Mouradian etal., J.Amer.Chem.Soc.118:8639-8645 (1996); Nelson et al., RapidCommun.Mass.Spectrom.9:1380-1385 (1995); Nelson et al., J.Monlec.Recognition 12:77-93 (1999).; Brockman et al., J.MassSpectrom 33:1141-1147 (1998); Yip et al., J.Biol.Chem.271:32835-33 (1996) also is applied to oligonucleotides, Jruinke et al., Anal.Chem.69:904-910 (1997); Tang et al., Nucl.Acids Res.23:3126-3131 (1995); Liu etal., Anal.Chem.67:3482-90 (1995), bacterium, Bundy et al., Anal.Chem.71:1460-1463 (1999), and micromolecule, Wei et al., the research of Nature 399:243-246 (1999).On business level, the affinity capture laser desorption ionisation be contained in Ciphergen protein-chip  system (Ciphergen Biosystems, Inc.Fremont, California, USA) in.

Although affinity capture laser ionization technology has solved a large amount of technical problems, also have difficulties.

When this method is applied to from biological sample capture protein, have only usually that a picomole of whole albumen is detected catches and can be used in subsequent analysis.Typically, the affinity capture on the chromatographic surface biochip does not cause purifying completely.In addition, viewed digestive efficiency in the sample of solid phase extractions is compared with the digestion of carrying out in free solution or two-dimentional gel sex change environment, and is relatively poor.Therefore, if about 50% be protein of interest matter, and successfully digested the about 10% of this albumen, have only about 50 certain peptides that fly mole to can be used in detection so at most.

In database digging experiment, use effective trypsase to digest the ox myosin, it is confirmed that, for example, even with the ultimate precision (an inaccessiable level of present most of MS technology) of 1.0ppm, when detecting the eukaryotic gene group of this complexity, can only obtain relatively poor protein ID coupling confidence level for the quality of single peptide.For two peptides, obtained lower confidence level result equally.Only after 3 peptides were provided, confidence level result just got back to the mass distribution that error is lower than 300ppm.In the case, most of equipment will need the internal standard verification.Yet under the situation of 5 or more peptide, the quality precision for being better than the 1000ppm error can not obtain further confidence level.

And, when numerous protein during, can produce uneven peptide pond, and successful database digging not only requires high precision, and the example of many elementary sequence informations will be arranged simultaneously by digestion.Series connection MS/MS method has been proved to be beneficial to very much elementary sequence information is provided.Biemann?et?al.，Acc.Chem.Res.27：370-378(1994)；Spengler?et?al.，RapidCommun.Mass?Spectrom.5：198-202(1991)；Spengler?et?al.，RapidCommun.Mass?Sepctrom.6：105-108(1992)；Yates?et?al.，Anal.Chem.67：1426-1436(1995)；Kaufman?et?al.，Rapid?Commun.Mass.Spectrom.7：902-910(1993)；Kaufman?et?al.，Intern.J.Mass?Spectrom.IonProcesses?131：355-385(1994)。

Yet up to date, unique MS/MS method that is used for based on the laser desorption analysis that can obtain is secondary (post) source decay analysis (PSD).Though PSD can provide considerable sequence information for the peptide of picomole level, the whole efficiency that this fragment is handled is lower; When combining with the relatively poor quality precision that often occurs and stability in the method, its application power that a small amount of protein of usually finding on affinity capture laser desorption ionisation probe is analyzed greatly is restricted.Recently, laser desorption ionisation four utmost point flight time mass spectrums (LDI Qq-TOF) grow up, and it can be carried out collision induced dissociation (CID) MS/MS and analyze.Krutchinksy?et?al.，Rapid?Commun.Mass?Spectrom.12：508-518(1998)。

Therefore, needs to the apparatus and method that can improve affinity capture laser desorption sensitivity of mass spectrometry and quality precision are arranged.Thereby have improving the needs that digestive efficiency on the probe allows to make the method and apparatus that the peptide that produces owing to the uneven protein mixture of digestion is easy to decompose.Needs to the apparatus and method that can improve affinity capture laser desorption tandem mass spectrum analysis efficiency are arranged.

Summary of the invention

An object of the present invention is to provide the apparatus and method of the sensitivity that can improve existing affinity capture laser desorption ionisation mass spectral analysis, quality precision, mass resolution, and improve the performance of MS/MS.Another object of the present invention provides utilizes these improved analytical performances to carry out biomolecule analysis.

In the first string, the present invention has satisfied purpose and needs such and such in the technology by a kind of analytical equipment is provided.

Analytical equipment of the present invention comprises, laser desorption ionisation source, affinity capture probe interface and tandem mass spectrometer, thereby wherein affinity capture probe interface can be meshed the affinity capture probe and it can be inquired about the probe location when getting in touch with tandem mass spectrometer by the laser desorption source, thereby allows ion to discharge and enter mass spectrometer from this probe.

Typically, the laser desorption ionisation source comprises laser-excitation source and laser light path system; This laser light path system plays the photon that will be excited is transmitted to the probe interface from laser-excitation source effect.In this embodiment, laser light path system typically transmits the energy of little joule of about 20-1000 to every square millimeter of surface by the inquiry probe.

Laser-excitation source is selected from the group that comprises continuous wave laser and pulse laser, and in various embodiments from comprising nitrogen laser, Nd:YAG laser, erbium: YAG laser and CO ₂Select in the group of laser.In this preferred embodiment, laser-excitation source is pulse nitrogen laser.

In one group of embodiment, laser light path system comprises the optics of selecting from the group that comprises lens, speculum, prism, attenuator and beam splitter.

In another group embodiment, laser light path system comprises an optical fiber with input and output, and the input coupling of laser-excitation source and this optical fiber connection.

In some optical-fiber laser light path system embodiment, laser light path system also comprises optical attenuator.This attenuator can be installed between laser-excitation source and the optic fibre input end, can play the effect that laser-excitation source is coupled to optic fibre input end, perhaps can be installed between fiber-optic output and the probe.

In some fiber optic system embodiment, fiber-optic output has the maximum gauge of about 200-400 μ m, and input has the diameter of about 400-1200 μ m.

This analytical equipment also can comprise probe viewing optics parts, with allow probe after mesh at itself and probe interface still as seen.

In certain embodiments, laser light path system can comprise laser couplers, and it is with laser-excitation source and optic fibre input end coupling connection.As mentioned above, this coupler can play the effect of optical attenuator.In other embodiments, coupler can play the effect that improves the probe visibility after probe and the engagement of probe interface.

In the embodiment of these back, coupler or optical fiber are branched and separate part energy from described laser-excitation sources.Selectively, thus this bifurcated can allow to import visible light illuminates the desorb site.

When comprising visual optics in the optical system, perhaps when the fibre-bearing laser light path system comprised two forks or trident, this analytical equipment can also comprise the light that fixation of C CD camera is returned from described probe reflection with detection.

In typical embodiment, affinity capture probe interface comprise can reversible engagement affinity capture probe probe holder.This interface also typically comprise itself can reversible engagement probe holder the probe introducing port.

In typical embodiment, the probe interface also comprises probe location governor assembly and interface ion gathering system.When probe holder and introducing port engagement, it is installed in and the contacted position of probe location adjuster; Conversely, this probe location adjuster can be with respect to laser ionization source (typically, with respect to laser light path system) and ion collection system position probe fixture (typical area the probe that it meshes) movably.In typical embodiment, this adjuster can the parallel or described probe holder of positioning of rotating.

The probe interface also typically comprises and the probe introducing port vacuum-evacuate system of coupling connection mutually, and this introducing port allows probe to be inquired about by the laser desorption ionisation source under pressure below atmospheric pressure.

Analytical equipment of the present invention comprises tandem mass spectrometer, and it is in various embodiments from comprising QqTOF MS, ion trap MS, and ion trap TOF MS selects in the group of TOF-TOF MS and Fourier transform ion cyclotron resonance MS.

In a preferred embodiment, tandem mass spectrometer is QqTOF MS and laser-excitation source is pulse nitrogen laser, the 2-4 that the laser current metric density at probe place is approximately minimum desorb threshold value doubly, and tandem mass spectrometer has the external perimysium reference quality precision of about 20-50ppm.

Analytical equipment of the present invention is designed to mesh with affinity capture laser desorption ionisation source.In addition, any the above embodiments can both comprise the affinity capture probe that meshes with affinity capture probe interface.

Affinity capture probe among these embodiment typically has at least one and is installed in the locational sample absorption surface that query relation is arranged with lasing light emitter, and the sample absorption surface is selected from the group that comprises chromatogram absorption surface and the affine surface of biomolecule.Typically, this chromatogram absorption surface is anti-phase from comprising, select the group of anion exchange, cation exchange, fixing metal affinity capture and mixed type surface, and the biomolecule on the affine surface of biomolecule is selected from the group that comprises antibody, acceptor, nucleic acid, lectin, enzyme, biotin, avidin, streptavidin, Staph albumin A and Staph Protein G.

Have on the position of query relation and the sample absorption surface of each self-routing but affinity capture laser desorption ionisation probe can have a plurality of can being placed on lasing light emitter, and can comprise at least two different this absorption surfaces.

In other embodiments, analytical equipment of the present invention comprises a digital computer that docks with the detector of tandem mass spectrometer.In certain embodiments, this equipment can also comprise the software program that can be carried out by this digital computer, the part of itself or this computer or can pass on access by this computer.Software program among these embodiment can be controlled the laser desorption ionisation source, perhaps control at least one aspect of tandem mass spectrometer data acquisition, perhaps carry out at least one analysis route, perhaps any one subclass of these functions of described tandem mass spectrometer data acquisition.

On the other hand, the invention provides the method that is used to analyze at least one detection albumen.

This method comprises that (a) catches the test proteins on the affinity capture protein bio-chip, (b) produces the protein catabolite that detects albumen with the decomposition of protein preparation on protein-biochips; (c) analyze at least one protein,split with tandem mass spectrometer.Among these embodiment of this scheme, analytical procedure comprises that (i) is discharged into protein,split the gas phase to produce corresponding parent ion peptide from protein-biochips, first mass spectrometer of (ii) selecting the parent ion peptide to be used for subsequently is cracked, (iii) under selected vapor-phase thermal cracking condition the selected parent ion peptide of cracking to produce the fragment ion product and (iv) to generate the mass spectrum of fragment ion product.In this mode, mass spectrum provides detecting the analysis of protein.

In some embodiment of this scheme of the present invention, this method also comprises additional step (d), by mass spectrum being submitted to Protein Data Bank digging agreement, it is based on close-fitting method between the theoretical mass spectrum of albumen in mass spectrum and the database is that detection albumen in the database is determined at least a protein candidate characteristic, determines at least a protein candidate characteristic for detecting albumen.

In the details of these embodiment, step (d) comprises that also quality and the original detection kinds of proteins that will detect albumen are submitted to this agreement.

In other embodiments, this method comprises that also (e) compares candidate's characteristic with detecting albumen, its be by: (i) produce the mass spectrum of protein cleavage product in (b); (ii) the mass spectrum with this protein cleavage product is submitted to computer protocol, it has determined the method that closely cooperates between the mass spectrum of the theoretical mass spectrum of the pyrolysis product candidate characteristic of prediction generating by using proteolysis reagent and protein cleavage product, whereby, this method shows that the protein cleavage product on the protein-biochips is consistent with detection albumen.

Yet also comprise step (f) among other embodiment of this method, repeating step (c) when selected parent ion peptide and the protein cleavage product of being expected by candidate's characteristic are inconsistent; And (g) repeats (d) to select the parent ion peptide in (f) then.

In this scheme of the present invention, detecting albumen can be the albumen of differential expression between first and second biological samples.In some such embodiment, first and second biological samples come from normal source and ill source.

In the 3rd scheme, the invention provides the method that characterizes binding interactions between the first and second molecule binding partners (binding partner).

In this scheme, this method comprises that in conjunction with second binding partners to first binding partners, wherein said first binding partners is fixed on the laser desorption ionisation detecting probe surface; Cracking second binding partners; Then survey at least one fragment with the tandem mass spectrum instrument measurement method, wherein be detected fragment mass spectral characteristi this binding interactions.

In some embodiment of this scheme of the present invention, before first binding partners, first binding partners at first is fixed in the surface of affinity capture probe in second binding partner binds.

This fixing can be by first binding partners directly be attached on the affinity capture probe, covalent bonding for example, and realizing.Typical covalent bonding embodiment comprises between the residue of the amine of first binding partners and described detecting probe surface carbonyl dimidazoles (carbonyldiimidazole), and the covalent bonding between the epoxide group of the amino of described first binding partners or thiol group and detecting probe surface.

Should be fixing also can be by direct non-covalent combination, the metal of first binding partners and detecting probe surface for example, as gold or platinum, between coordination or complexing combination and realizing.The chromatogram absorption surface of should be fixing selecting can also the group by first binding partners and, anion exchange anti-phase from comprising, cation exchange fixing metal affinity capture and mixed type surface interacts and realizes.

Selectively, this is fixing can be indirect, though be indirect also can be covalency.In some embodiment of back, first binding partners can adopt covalent bond and by linker, for example the linker of cleavable and fixing.Indirect securement also can be non-covalent, for example by biotin/avidin, and the interaction of biotin/streptavidin and being fixed on the probe.

In this scheme of the present invention, the first molecule binding partners can be selected from the group that comprises protein, nucleic acid, carbohydrate and lipid.Typically, first binding partners is a protein, it can be a spontaneous protein in the organism of selecting from the group that comprises many cells eucaryote, unicellular eukaryote, prokaryotes and virus, perhaps can the spontaneous protein of right and wrong, for example recombinant fusion protein.

At first binding partners is among the embodiment of protein, and this albumen can be selected from the group that comprises the antibody, acceptor, transcription factor, cytoskeletal protein, cyclin and the ribosomal protein that are mixed in other albumen.

Second binding partners combines with first binding partners of fixing, in typical embodiment, is subjected to the contacted influence of first binding partners and biological sample; This sample can be the fluid of selecting from the group that comprises blood, lymph liquid, urine, celiolymph, secretion synovia, milk, saliva, vitreous humor, watery body fluid, mucus and seminal fluid, or cell lysates, the perhaps sample of some other form.

In various embodiments, comprise that first binding partners is the embodiment of protein, second binding partners can be a protein.Selectively, second binding partners also can be the compound that is present in the combinatorial libraries, and wherein second binding partners is subjected to first binding partners and the contacted influence of part chemical synthesis combinatorial libraries with combining of first binding partners.Yet in another was selected, second binding partners can be the biological component of Henan combinatorial libraries late, for example slow mutually storehouse, Henan.

In some typical embodiment, cracking is subjected to second binding partners and the contacted influence of enzyme; When second binding partners was protein, enzyme was typically special integrated protein enzyme, for example insulin, Glu-C (V8) protease, inner protease Arg-C (cysteine proteinase), Asn-N protease and Lys-C protease.Selectively, cracking can be subjected to described second binding partners and liquid phase chemical reagent, CNBr for example, contacted influence.

In certain embodiments, this method also comprises, after second binding partners and described first binding partner binds, before cracking second binding partners, makes the second binding partners sex change.

In various embodiments, this method also comprises step, after the second binding partners cracking, use first eluent, sometimes use second eluent, the flushing probe, second eluent is having difference by a wash-out characteristic and first eluent at least, for example pH, ionic strength, decontamination intensity and hydrophobicity.

In typical embodiment, cracking after and before the detection second binding partners fragment, this method also comprises step, and the energy absorption molecule is applied to probe.In a preferred embodiment, probe meshes with the affinity capture probe interface of analytical equipment of the present invention subsequently, and discharges with the fragment ionization of second binding partners and from probe with the lasing light emitter of this equipment.

This equipment can comprise the method for measuring all mass of ions with the multiple effective method of measurement that generates this method, measures the method for quality of a part of fragment, and the method for supervising of single ionic.

Effectively, the embodiment of this method comprises step: after the second binding partners fragment is by mass-spectrometer measurement, the fragment measurement result is compared with the prediction of making to the initial amino acid sequence of second binding partners by application lyases cracking rule, whereby, thisly more just characterized out intermolecular interaction.

If characteristic the unknown of second binding partners, this method can also comprise, before this comparison, analyze to determine the characteristic of second binding partners by MS/MS.This MS/MS analyzes can comprise step, and first fragment of second binding partners is selected on mass spectroscopy ground; First fragment of cracking second binding partners in gas phase is then compared the fragment frequency spectrum with the fragment frequency spectrum that dopes from the amino acid sequence data that adds in advance the database.Amino acid sequence data can be selected from the group that comprises experimental data and pre-primary data, and this dissociates, and in typical embodiment, is collision induced dissociation.

In some embodiment of this method, first binding partners is selected from the group that comprises antibody, TXi Baoshouti and MHC molecule.In other embodiments, to be acceptor and second binding partners select from the group of the part antagonist of the antagonist of the antagonist that comprises this receptor, this receptor partial antagonist, this receptor and this receptor first binding partners.In other embodiments, first binding partners is that the glycoprotein receptor and second binding partners are lectins.

In the 4th scheme, the invention provides the method for detecting analytes, this method comprises the affinity capture probe is meshed with the affinity capture probe interface of analytical equipment of the present invention that this affinity capture probe also has the limiter of analyte; Lasing light emitter with this equipment discharges analyte or fragment or ionization from probe; Then measure d/d ion and survey this analyte by tandem mass spectrometer.

In this scheme, this method can also comprise, after desorb and ionization steps and before surveying, realizes the collision induced dissociation of this release ion.Before this dissociating, a part of in certain embodiments ion can choose to be used for collision and dissociate.

In other embodiments, can carry out analyte is adsorbed in first step on the probe, and in a further embodiment, after the adsorption analysis thing and before this probe and the engagement of this probe interface, can carry out the step that this probe is contacted with this analyte adhesion with the energy absorption molecule.

Description of drawings

Purpose that reaches other that the present invention is above-mentioned and advantage will be apparent by the following detailed description taken in conjunction with the accompanying drawings of reference, and similar symbol is represented similar part from start to finish in the accompanying drawing, wherein:

Fig. 1 has represented an embodiment of analytical equipment of the present invention in a capsule;

Fig. 2 has showed the element of the preferred quadrature QqTOF tandem mass spectrometer that uses in the analytical equipment of the present invention in more detail;

Fig. 3 has shown the spermatin matter profile of single BPH and patients with prostate cancer;

Fig. 4 has showed the result who separates on (upregulated) albumen probe of detectable rise among a kind of Fig. 3;

Fig. 5 has showed after enriched biological label material standed for is exposed and carries out original position digestion with insulin, analyzes the peptide of detection with single phase MS;

Fig. 6 has showed that the LDIQq-TOF MS of identical protein purification peptide on analytical equipment of the present invention analyzes; With

Fig. 7 has showed the MS/MS result that two times of charged ions of selected enriched biological label material standed for obtain from analytical equipment of the present invention.

Embodiment

I. definition

As used herein, the term of listing especially below has following definition.If not definition in addition, all terms of Shi Yonging have the implication of professional person institute common sense in the technical field of the invention herein.

" analyte " is meant any component of the sample that hope is detected.This term can relate to one-component or a plurality of component in this sample.

" probe " is meant such device, when being positioned engagement and when having query relation with the laser desorption ionisation source and under atmospheric pressure or pressure below atmospheric pressure, keeping parallel contact, can be used in the ion that guiding comes from analyte and enter mass spectrometer with the gaseous ion mass spectrometer.As used herein, should " probe " typically with the reversible engagement in probe interface.

" affinity capture probe " is meant by being enough to allow the interaction of extracting and concentrating analysis from heterogeneous mixture of this probe to come the probe of bound analyte.The purity that concentrates does not also require.The probe absorption surface mediates this binding interactions by analyte is adsorbed onto typically.Term protein chip (ProteinChip)  array be meant used in the present invention can be commercial from Ciphergen biosystem Co., Ltd, Fremont, California, the affinity capture probe of acquisition.

" absorption " is meant the detectable non-covalent combination of analyte to adsorbent.

" adsorbent " is meant any material that can the adsorption analysis thing.The term of Shi Yonging " adsorbent " had both related to single material (" monobasic adsorbent ") (for example, a kind of compound or a kind of functional group) and had also related to multiple different material (" polynary adsorbent ") herein.Sorbing material in the polynary adsorbent relates to as " adsorbent material ".For example, the laser addressed absorption surface on the probe matrix can comprise that with many different adsorbent material (for example, anion-exchange material, metal-chelator or antibody) with different binding characteristics be the polynary adsorbent of feature.

" absorption surface " is meant the surface with adsorbent.

" chromatogram absorption surface " be meant have can chromatogram the surface of adsorbent of identification or separate analytes.Therefore this term comprise have anion exchange part, cation exchange part, anti-phase part, metal affinity capture partly and the surface of mixed type adsorbent, as those in the chromatographic technique field the term that can understand.

" the affine surface of biomolecule " is meant to have the surface that comprises biomolecule that can specific bond.

" specific bond " be meant two kinds be present in simultaneously in inhomogeneous (non-homogeneous) sample molecular species with respect to sample in other molecular species combine the ability that is more prone to combination each other.Typically, specific bond interacts than the recognition capability height of accidental binding interactions in reaction twice at least, more typically surpasses 10-100 doubly.In the time can determining that analyte is present in inhomogeneous (non-homogeneous) sample, when being used for detecting analytes, specific bond is enough to be used in identification.Typically, the affinity of specific binding reaction or affinity about at least 10 ^-7M typically has at least 10 in having bigger specific specific binding reaction ^-8M is to about at least 10 ^-9The affinity of M or affinity.

" energy absorption molecule " and corresponding initial " EAM " be meant can, in the time of on adhering to probe, absorb energy from the laser desorption ionisation source and promote the analyte desorption of contact with it and the molecule of ionization subsequently.This word is included in United States Patent(USP) Nos. 5,719, and 060,5,894,063,6,020,208 and 6,027, all molecules described in 942, its disclosed content is comprising its whole list of references.This word obviously comprises styrene acid derivative, sinapine acid (" SPA "), cyanogen hydroxy styrenes acid (" CHCA ") and resorcylic acid.

" tandem mass spectrometer " is meant any gaseous ion mass spectrometer, and it can carry out the identification based on m/z of two successive stages to the ion in the ion mixture.This word comprise mass spectrometer with two mass-synchrometers and have can be before quality analysis selectivity obtain or keep the mass spectrometer of the single mass-synchrometer of ion.Therefore this word obviously comprises QqTOF mass spectrometer, ion trap mass spectrometer, ion trap-TOF mass spectrometer, TOF-TOF mass spectrometer and Fourier transform ion cyclotron resonant mode spectrometer.

" eluent " is meant reagent, solution typically, and it is used to influence or regulates the absorption of analyte to the absorption surface adsorbent.Eluent also relates to as " agent of selectivity threshold value adjustment " herein.

" wash-out characteristic " is meant the physics or the chemical characteristic of eluent, and it helps its influence or regulates analyte to the adsorbent ability of absorption surface.If when analyte was contacted with eluant, eluent, analyte was to the affine degree difference of eluant, eluent, then two kinds of eluant, eluents have different wash-out characteristics.The wash-out characteristic comprises, for example, and pH, ionic strength, unordered degree, decontamination intensity and temperature.

" biological sample " and " biological sample " all is meant the sample that comes from the organism at least a portion that can duplicate.As used herein, biological sample can come from any known taxology field, comprises virus, prokaryotes, unicellular eukaryote and many cells eucaryote.Biological sample can come from organic integral body or its part, comprises that coming from it cultivates part.Biological sample can be in and be suitable for contextual any physical form, comprises homogenate, subcellular components, lysate and fluid.

" biomolecule " be meant and can find in biological sample, but do not need to come from the organic molecule of biological sample, for example steroids, amino acid, nucleic acid, sugar, polypeptide, polynucleotide, complex carbohydrate and lipid.

" organic molecule " is meant the organic molecule that size can be compared with the organic molecule that those use usually in medicine.This term does not comprise organic biopolymer (for example, protein, nucleic acid or the like).Herein the organic molecule of Shi Yonging typically size range be up to about 5000Da, up to about 2500Da, up to about 2000Da or up to about 1000Da.

" biopolymer " be meant and can find in biological sample, but do not need to come from the polymer of biological sample, for example polypeptide, polynucleotide, polysaccharide and polyglycerol ester (for example, two or glyceryl ester).

" fragment " is meant chemistry, enzyme or the physics breakdown products of analyte.Fragment can be in neutrality or ionic condition.

Term " polypeptide ", " peptide " and " protein " that can exchange use herein is meant that abiogenous or synthetic polymer comprises amino acid monomer (residue), wherein amino acid monomer herein comprises abiogenous amino acid, abiogenous amino acid structure variant and can participate in the synthetic non-natural occurring analog of peptide bond.Polypeptide can be conditioned, and for example forms glycoprotein by additional carbohydrate residue.Term " polypeptide ", " peptide " and " protein " comprise glycoprotein and non-glycoprotein.

" polynucleotide " and " nucleic acid " is meant that all abiogenous or synthetic polymer comprises nucleotide monomer (base).Polynucleotide comprises abiogenous nucleic acid, for example DNA (deoxyribonucleic acid) (" DNA ") and ribonucleic acid (" RNA "), and nucleic acid analog.Nucleic acid analog comprises that those comprise the analog of non-abiogenous base, is not the nucleotide monomer that is connected with abiogenous phosphodiester bond with those.Nucleotide analog comprises, for example (and being not limited thereto) phosphorous acid thioesters (phosphorothioates), phosphorous acid dithioesters (phosphorodithioates), phosphotriester, phosphoric acid branch hydrochlorate (phosphoramidates), phosphoric acid borine, methyl phosphorodithioate, chirality methyl phosphate, 2-oxygen-methyl ribonucleotides, peptide-nucleic acid (PNAs), and analog.

As used herein, " molecule binding partners "---and comparably, " specific bond gametophyte "---be meant that the molecule that shows specific bond is right, biomolecule is right typically.Example that is not limited thereto such as acceptor and part, antibody and antigen, biotin and avidin, and biotin and streptavidin.

" acceptor " is meant molecule, big typically molecule, and it can find in biological sample, but does not need to come from biological sample, and it can participate in the specific bond with part.This term also comprises fragment and the derivative that keeps the sepcific ligands binding ability.

" part " is meant the acceptor that can participate in and design or any component of antibody specific bond.

" antibody " is meant that basically by the fragment of at least one immunoglobulin gene encoded polypeptides or at least one immunoglobulin gene, it can participate in the specific bond with part.This term comprises abiogenous form, and fragment and derivative.Fragment in the employed herein term field comprises that those are used various peptases, for example Fab, Fab ' and F (ab) ' 2 fragment digests and the fragment of generation, the fragment that those produce by chemical breakdown, by the cracked fragment that produces of chemistry, and the fragment of reorganization, as long as this fragment keeps and the ability of target molecule specific bond.Typical reorganization fragment as producing by for example bacteriophage video picture, comprises strand Fab and scFv (" strand Variable Area ") fragment.Derivative in this term field comprises antibody (or its fragment), and its sequence is conditioned, but still can the specific bond target molecule, chimera and people's antibody between comprising kind.As used herein, antibody is to comprise by the bacteriophage video picture by any known technology, perhaps cutting, hybrid cell, the recombinant expression system of similar techniques acquisition from the lymphocytic cell of natural B is cultivated.

" antigen " is meant can be by the part of antibody combination.Antigen needs not be immunogene.The contacted part of antigen and antibody is named as " antigenic determinant ".

" flux density (Fluence) " is meant the energy that is delivered to by on the inquiry video per unit area.

II. affinity capture probe tandem mass spectrometer

In the first string, the invention provides a kind of analytical equipment, it combines the advantage of the advantage of affinity capture laser desorption ionisation sample introduction and tandem mass spectrometer high accuracy, high-quality resolution rate.This combination is used to carry out the known technology apparatus operating and has sizable advantage than existing.And this new equipment makes the new method of finding protein become possibility, also makes the identification of interaction of molecules between the specific bond gametophyte and sign, and the new method rapider, more effective, sensitiveer than existing method becomes possibility.This equipment is once briefly described at first earlier generally; Again illustrate in greater detail the characteristic at this affinity capture probe interface thereafter.

Briefly, with reference to figure 1, equipment 100 comprises laser desorption/ionization source 13; Affinity capture probe interface 10 and tandem mass spectrometer 14.Shown in Figure 1 is a preferred embodiment, and wherein lasing light emitter 12 is pulse nitrogen lasers, and tandem mass spectrometer 14 is quadrature four utmost point time-of-flight mass spectrometers (QqTOF) series connection MS.

Laser desorption/ionization source

Laser desorption/ionization source 13 produces high-energy photons, is suitably regulated and guides, and its desorb and ionization are adhered to protein and other analyte of affinity capture probe 16.Laser desorption/ionization source 13 comprises lasing light emitter 12, laser light path system 11 and, selectively, and probe viewing optics parts 18.

Laser desorption/ionization source 13 perhaps, as selection, produces pulsed laser energy by mechanically or electronically cutting the light beam that transmits from continuous wave laser 12 by using pulse laser 12.Typically, pulse laser is preferential the selection.Preferred pulsed laser source comprises nitrogen laser, Nd:YAG laser, erbium: YAG laser and CO ₂Laser.Pulse nitrogen laser preferably herein because its take up an area of simple and cost relatively low.

The photon that sends from laser 12 is directed clashing into probe 16 by laser light path system 11 surface.Optical system 11 can comprise a configuration of being made up of lens, speculum, prism, attenuator and/or light beam dispenser, it plays collection, guiding, focusing, separates and controls the effect that each restraints laser pulse intensity, thereby the desorb energy that will be in the suitable desorb flux density of having of focus point form is delivered on the probe 16.

Selectively, optical system 11 can comprise a fiber array, the effect that it plays collection, guiding and separates each bundle pulsed laser energy again

In this embodiment, the output of laser 12 and the input of optical fiber join with the optical coupler coupling; This coupler typically comprises the lens that a focal length and diameter all are suitable for the optic fibre input end numerical aperture.

The amount of energy that enters optical fiber can be by careful adjusting lens with respect to the position of fiber and controlled; In this example, the optical coupler of fiber can also be as optical attenuator.In another preferred disposition, the whole output energy of laser all is imported in the fiber and attenuator places between fiber-optic output and the optical system desorb point focusing element.In the another one preferred disposition, optical attenuator places between laser and the fiber coupler.In all examples, optical attenuator all is to be used for guaranteeing to transmit the surface of suitable laser current metric density to probe 16, and is independent of the output energy of laser 12.Typical laser current metric density is in the magnitude of little Jiao of 20-1000/square millimeter.

Such just as confirmed, when receiving the energy of the focusing of transmitting from laser, the fiberoptics parts may usually be damaged, so be favourable with the maximization of the receiving area of fiber input, the flux density of the laser energy that imports into like this is lower than the damaging thresholding of fiber.The latter has also simplified when adjusting optical coupler with respect to laser and fibre-optic position the calibration to fiber laser beam.Yet in order to obtain quite high desorb flux density level on probe 16, when to use typical nitrogen laser to transmit ceiling capacity be about 200 μ J/ laser pulses, maximum exit fibre diameter should not surpass 400 μ m (micron).The solution of this problem is to introduce the tapered optical fiber, and its input has the diameter of 400-1200 micron dimension and output has the diameter of 200-400 micron.

Typically, the desorb point should focus on the maximized size of ion that each beam pulse is produced, and it is by when maintenance is enough to bring out the flux density of desorb and ionization, and maximum probe 16 areas of inquiry are realized.Join in the laser desorption/ionization source of four utmost points-four utmost point flight time tandem mass spectrometer at coupling, when using the ceiling capacity of the about 200 little Jiao/pulses of typical nitrogen laser transmission, optimum laser spots area has been determined to be in the scope of 0.2-0.4 square millimeter.

Laser desorption/ionization source 13 can comprise, and the typical case is as the part of optical system 11, probe viewing optics parts 18.Viewing optics parts 18 can comprise light source, lens, speculum, prism, dichroic reflector, band pass filter and CCD camera to allow illumination and observation desorb point, for example, and the zone that probe 6 is inquired about by laser.

When laser light path system 11 comprises optical fiber, the light that viewing optics parts 18 can utilize optical fiber itself to send.

For example, fiber optic coupler can be divided into two parts (being bifurcated) to separate a fraction of laser excitation energy, thereby as a kind of method that is used for monitoring the laser energy that is applied, perhaps it can be divided into two parts and illuminates the desorb point allow to introduce visible light.

In first embodiment of these two embodiment, a fraction of excitation energy be directed clashing into as laser energy loop part take the photograph the phase detector, its through calibration can the effective quantity of reflected back the laser energy that passes to probe 16.In second embodiment, visible light is directed illuminating desorb point and makes that observing this zone becomes possibility, it is or takes the photograph the phase optics by one group of independent coupling connection CCD camera, perhaps by between optical fiber and laser-excitation source, using prism or dichronic mirror, its light that will reflex to the optical fiber main road is directed on the CCD camera, and realize.Selectively, prism or dichronic mirror can place between optical fiber illuminated fibres branch road and the light source as the crow flies to allow any image that reflects that is coupled to this branch road to be directed clashing into the CCD camera.In another embodiment, this fiber can be divided into trident, thereby one is transmitted desorb/ionization laser pulse, another transmission be used to throw light on visible light of desorb point, and the 3rd will be submitted to the CCD camera from the light that the desorb point reflection is returned.For each observation plan in the middle of these, suitable band pass filter should be arranged between CCD camera and the viewing optical system, be delivered to detecting probe surface with the destructive high-energy photon of avoiding to take place along with the direct reflection of the laser pulse that imports into that may have, perhaps avoid the secondary photon to overflow from detecting probe surface as the direct result of the electron excitation of the laser pulse that imported into.

The probe interface

Affinity capture probe interface 10 can reversible engagement affinity capture probe 16, and position probe 16 makes it to have query relation with lasing light emitter 12, and keeps in touch with tandem mass spectrometer 14 simultaneously; This contact can be supported to be pressed onto pressure below atmospheric pressure from atmosphere.

Probe interface 10 comprises that probe holder, probe introducing port, probe location governor assembly, vacuum and Pneumatic assembly put, and the interface ion gathering system.

Probe holder is the parts at probe interface 10, and it is formalized so that consistent with the form factor (form factor) of probe 16.When probe 16 was protein-chip  array (Ciphergen biosystem Co., Ltd, Fremont, CA USA), probe holder was consistent with the form factor of protein-chip  array.

Probe holder can fixed single probe 16 or a plurality of probe 16.Fixture is positioned each probe 16 for the orientation suitable with respect to the ion collection system interface, to be inquired about by laser desorption/ionization source 13.

Probe holder closely contacts with the position control assembly.

This governor assembly can be with respect to the relative position of laser desorption/ionization source 13 and ion collection system Interface Moving probe 16, thereby the zones of different of probe can both be inquired about and the ion that produces owing to this irradiation is collected to be incorporated in the tandem mass spectrometer 14.

This adjuster is included in the electrochemical appliance of supporting when the position that makes probe keeps constant with respect to the position of laser desorption/ionization source and ion collection system that probe 16 is parallel and/or rotation is moved.This electrochemical appliance includes but not limited to: machinery or optic position sensor, solenoid, stepper motor, DC or AC syncmotor, its directly or indirectly with linear movement governor, linearity or circumference moving guide rail, universal joint, bearing, or axostylus axostyle is communicated with.

The probe introducing port allows probe holder, comprises the probe 16 that is loaded, and places on the probe location governor assembly and the atmospheric gas of not introducing incorrect level enters probe interface 10 and tandem mass spectrometer 14.

In order to realize the latter, the probe introducing port has used vacuum-evacuate system (probe introducing port evacuation system) to pump atmospheric gas, obtains the target port pressure before chip is moved into the service position.Between the probe commutation period, (with laser desorption source 13 and ion collection system position in line) moves to switch to the Probe Regulator assembly from the service position with probe.In such operation, adjuster can provide sealing soon rising to rapidly between atmospheric exchange mouthful and the mass spectrometer input.After having sealed the mass spectrometer input, atmospheric gas is imported in the probe introducing port by probe introducing port compression system.This has just eliminated the pressure differential between probe holder atmosphere surface and the introducing port, allows probe holder to remove from the probe location adjusting part.

Be accompanied by removing and the installation of new probe 16 of previous analysis probe 16, probe holder is taken back to the position that its position control and sample loading process begin.As previously mentioned, the probe introducing port can enough evacuation system be drawn into pressure below atmospheric pressure.In case arrive the importing pressure of target sample, the probe regulating system just moves to the service position with probe 16 from switch, and opens the sealing to the mass spectrometer input in this operates.

Selectively, produce and when finally being directed on the ion optics when ion is being in the atmospheric desorption chamber, it to the mass spectrometer input, does not need ion guides the probe introducing port is found time and pressurizeed, because it is held under atmospheric pressure.

Probe introducing port evacuation system comprises vacuum pump, pressure sensor, vacuum compatible conduit and connection fittings, and the compatible valve of vacuum, when co-operation, when it allows to be accompanied by the sample exchange atmospheric gas that is retained in the introducing port is carried out controlled finding time, thereby probe 16 can be moved to the service position.Vacuum pump can be (but being not restricted to this) one pole or multipole oily mechanical pump, scroll pump, or oil-free diaphragm pump.In a preferred embodiment, the compatible valve of vacuum is automatically controlled solenoid valve.In identical embodiment, pressure sensor is can be at the electronic sensor of the pressure limit work that is pressed onto 1 millitorr from atmosphere.This pressure sensor comprises (but being not limited thereto) thermocouple needle and pirani meter.In identical embodiment, this system's same operation is to realize under the logic control that is provided by analog logic loop or digital microprocessor, and wherein the input one of analog logic loop or digital microprocessor and pressure gauge and position transducer is shown permission and found time automatically as the sample port of a whole system operation part.

Probe introducing port compression system comprises gas source, pressure sensor, gas introduction tube road or accessory and the compatible valve of gas, and when co-operation, it allows the gas of controlled importing to exchange mouthful pressurization, thereby allows probe holder is removed from adjusting part.

In one embodiment, gas source is undressed atmospheric gas.In another embodiment, gas source is at first to be conducted through the water absorbing agent trap, and selectively passes through the atmospheric gas of special filter before importing compression system again.In another embodiment, be to provide using the occasion of atmospheric gas, pressurization gas by the purifying source of forming by dry inactive gas such as nitrogen or any cost-efficient inert gas.

In a preferred embodiment, the gas introduction tube road of compression system, accessory, some valve and pressure sensor are employed in evacuation system.In identical embodiment, this system's identical operations is to realize under the logic control that is provided by analog logic loop or digital microprocessor, and wherein analog logic loop or digital microprocessor utilize the input of pressure gauge and position transducer to pressurize automatically with the sample port that allows to operate a part as whole system.

Probe interfacial pressure control system has played being arranged in probe 16 samples and has existed desorption chamber between (absorption) surface and the ion collection system that the effect of selectivity background gas pressure is provided.Acceptable desorption chamber pressure limit from atmospheric pressure until 0.1 microtorr.The preferred pressure scope is 1 to hold in the palm 1 millitorr.Probe interfacial pressure control system comprises gas source, gas introduction tube road and accessory, gas flow controller and pressure sensor.Gas source can be undressed atmospheric gas.In another embodiment, gas source is the atmospheric gas that at first is conducted through the water absorbing agent trap and selectively passed through special filter again before importing control system.In another embodiment, control gaseous is to provide by the purifying source of being made up of dry inactive gas such as nitrogen or any cost-efficient inert gas.Gas flow controller can be the flow restrictor of manually controlling.Selectively, gas flow control can realize by using the Electronic Control flow restrictor.In a preferred embodiment, the closed-loop control of preferred desorption chamber pressure is under the logic control that is provided by analog logic loop or digital microprocessor, realize that with automatic pattern wherein analog logic loop or digital microprocessor interact with the reading that obtains from pressure gauge to establish in advance with the automatic gas flow controller delicately.

The interface ion gathering system comprises electrostatic ionic collection assembly, optional aerodynamic ion collection assembly and static or RF ion guiding device.The electrostatic ionic collection assembly comprises DC electrostatic lens arrangements of components, and it plays the ion that is collected in desorb in the desorption chamber and it is directed to the effect of mass spectrometer input.

In one embodiment, this assembly comprises two electrostatic elements.First element comprises probe holder and detecting probe surface, and second is dialyte lens.Dialyte lens is placed on from array surface 0.2-4mm place far away.Dialyte lens comprises the aperture that diameter range is 2-20mm, and it is placed with one heart around the normal axis that extends to mass spectrometer input center from desorb dot center.Independently the DC electromotive force is applied to each element of this assembly.

In a preferred embodiment, dialyte lens comprises the aperture of diameter 10mm and is placed on 1mm place far away, distance arrays surface.In identical embodiment, 10 volts of electrical potential differences are set up between separator and array.

The aerodynamic ion collection assembly comprises gas source, guiding pipeline, baroceptor and gas escape orifice, moves in the mass spectrometer input to help the maldi ion integral body in the desorption chamber thereby can produce predetermined gas flow.

Gas source can be undressed atmospheric gas.In another embodiment, gas source is at first to be conducted through the water absorbing agent trap atmospheric gas by particular filter selectively before import system again.In another embodiment, ion collection gas is to provide by the purifying source of being made up of dry inactive gas such as nitrogen or any cost-efficient inert gas.

Gas flow controller can be the flow restrictor of manually controlling.Selectively, air-flow control can realize by using the Electronic Control flow restrictor.Pressure sensor can be (but being not limited thereto) thermocouple needle and priani meter.Gas vent places probe 16 back to flow and flow to the normal axis that is positioned over desorb point and mass spectrometer input with one heart around probe with guide main body gas.

In a preferred embodiment, gas flow is subjected to automatic loop control by using the analog or digital control circuit, thereby has produced enough ion scan (sweeping) flow and inexcessive pressing and desorption chamber.

Last parts of interface ion gathering system are ion guiding devices.Ion guiding device has played the effect of collected ion transport to mass spectrometer 14.It can belong to static or RF kind.Preferred embodiment is multipole RF ion guiding device.The latter's a example is four utmost points or sextupole ion conduit.In the preferred Qq-TOF equipment that is described in more detail below, the ion conduit is four utmost point RF ion conduits.Ion is entered ion guiding device by the static or the guiding of pneumatic accelerative force that are produced by static and pneumatic ion collection system respectively.The DC electrostatic potential score of ion guiding device in a preferred embodiment is from the typically little 10-20 volt of lens.

Tandem mass spectrometer

Analytical equipment of the present invention also comprises tandem mass spectrometer 14.Tandem mass spectrometer 14 can be selected from the group that comprises four utmost point flight time of quadrature (Qq-TOF), ion trap (IT), ion trap flight time (IT-TOF), flight time-flight time (TOF-TOF) and ion cyclotron resonance types such as (ICR) effectively.

Preferred herein, and further specify below, be quadrature Qq-TOF MS.

The main strength of QqTOF MS is superior quality precision and resolution capability; The peptide sensitivity and the low mw scope that improve; And good ms/ms performance by adopting low energy collision induced dissociation (CID) to produce.Quadrature QqTOF with electron spray ionisation source can be from AB/MDS Sciex (QSTAR ^TMAB/MDS-Sciex, Foster City, California is USA) in commercial acquisition.

With reference to figure 2, principle and the characteristics of QqTOF have been summarized briefly.

Ion produces in first quadrupole lens " q0 " desorption chamber before.Pressure among the q0 typically maintains about 0.01-1 holder, but also can keep under atmospheric pressure.In this method, maldi ion after it forms soon just by with the background gas collision and cool off rapidly.

The integral body cooling or the decay of this ion provide three main advantages.

At first, the cooling initial energy of having eliminated maldi ion distributes and its gross energy is reduced to degree near its heat energy.This has simplified the requirement that quadrature separates, and has remedied the different of ion position and energy, has therefore improved final resolution capability.The direct result that resolution is improved is that the quality precision has been brought up to lower ppm level.

Second advantage of collision cooling is that it can reduce the speed of long-term ion decay.Gas collisions has relaxed internal motivation and has improved peptide and the stability of protein ion.When ion pressure be approximately 1 holder background gas in the presence of when producing, as if this stabilization effect be maximized.Other people disclosed measurement shows loss and the background fragment that can eliminate little basic group effectively, has improved the transmission of high mw protein and other unstable biopolymer (glycerine conjugate for example, DNA, or the like).The mechanism that decays faster (type decay in instant type and the source) also can take place.

Last advantage of q0 collision cooling is to produce the pseudo-continuous ionic stream that enters mass analyzer.Ion collision among the q0 causes that the desorb cloud launches along the axis of q0.This expansion has produced a kind of situation, and promptly wherein various desorption processes begin overlapping, have produced the continuous guiding that the ion of similar electron spray is entered analyzer.

After q0, ion enters into second four utmost point 22 (" Q1 ").This four utmost point has played or as the ion conduit or as the effect of mass filter.Having produced the ion that is used for ms/ms or single ionic supervision (SIM) experiment here selects.

Leave after the Q1, ion enters into the 3rd four utmost point 24 (" q2 ") that is arranged in collision cell 26.During simple experiment, q2 exercises the function of simple rf ion conduit.For the ms/ms experiment, q2 is with about 10 ^-2The collision gas of holder is filled to promote low energy CID.

Leave after the q2, ion quickens slightly by the DC electrical potential difference that is applied between q2 outlet and the focus mask 28.This acceleration " deflection " is along the ion velocity of Y-axis, thereby the square root of its speed and its m/z is inversely proportional to herein.If whole ions with different m/z is all wanted collision detector after quadrature separation and free flight, just must realize this deflection.If this deflection does not realize that the ion of different m/z will enter the quadrature separated region with identical Y-axis speed.

As in the flight time exist always, have the ion of low m/z can be before the ion with big m/z collision detector.Absolute displacement level along Y-axis has produced ion along the flight time of Z axle and the Y-axis speed of ion.If detector is placed on some for intermediate state mw ion on the optimized position, then lighter ion will " undershoot " detector, arrive the right side of detector among Fig. 2.On the contrary, has the left side that " shooting surpasses " detector is arrived detector among Fig. 2 than the ion of big m/z.As a result, if all ions all clash into same detector site, just be necessary to make all ions all to keep constant Z axle and Y-axis velocity rate.Aforesaid grid deflection method has just realized this purpose.

After focus mask 28, ion has arrived the regulator region 30 of quadrature resolution element.Adjuster 30 is with the adjusting of being pulsed near the speed of 10,000 pulse/sec (10KHz).Ion is pushed in the accelerator post 32 of ion-optic system, then leaves the free flight zone 34 that it enters into the quadrature flight time (O-TOF).When ion entered ion scintilloscope 36, energy correction had just been finished.In scintilloscope, ion is diverted and is directed to clash into the micro-channel plate detector 38 of V-arrangement arrangement and produces fast response.

But other system of selection of this prototype configuration also can be adopted.

For example, the geometry of carrying has above brought the difficulty of carrying out O-TOF under high acceleration energy.Determine preferably, the sensitivity of ion detection peptide and protein improves along with the rising of all ion energies.For actrapid monotard (MW=5807.65Da), when using typical micro-channel plate detector, under the ion energy of 35keV, detection efficient is near 100%.Have 20 or the energy of 30keV if ion is accelerated to, then free flight pipelining 40 and other associated components just must float to respectively-20kV or-30kV.Under this electromotive force, it is well-known that the difficulty of stable electrical insulation is provided on the simple ion optical element.A plurality of elements that float safely and reliably under this electromotive force are difficult.A solution adopts the post acceleration technology exactly.

Different with said apparatus, this alternative device has adopted detector postaccelerator (not shown).Ion is accelerated to the energy with about 4keV after leaving the quadrature resolution element, and the free flight zone is floated to-4kV.Along with entering the postaccelerator detector assembly, ion just realized further acceleration.In this assembly, ion is kept grid through the field that remains under the lining electromotive force.On the scene the keeping in the field of setting up between grid and the detector primary ion conversion surface of ion quickened again then.This accelerating field is in the magnitude of 10-20kV, and span is 4-10mm.

Because orthogonal design is separated the measurement of flight time with the formation of ion, many advantages have been realized.

Laser current metric density relevant issues, the peak broadening that produces owing to ion shielding and the stack of ion accelerating field for example can be because the desorb plume of ion stream have the longer time (typically being several milliseconds) to expand and cool off and be eliminated before quadrature separation and acceleration enter the TOF mass analyzer.In addition, quadrature separates has eliminated a large amount of big peak and baseline abnormal, can be observed owing to the chemical noise that the neutral load of too much EAM be produced when its high laser energy at tradition separation wave spectrum begins.Because neutral particle is not separated in regulator region, have only ion to be passed in the detector, thereby chemical noise has considerably been reduced.

These factors can allow to use the laser current metric density doubly than common employed big 2-3 in the ion isolation method of parallel continuous or slow Henan.Its final result is, even under the situation of relatively poor sample-EAM homogeneity, the needs of following the tracks of and search for " scanning element " have almost completely been eliminated, also improved determine (typical error is 20-50ppm) of external perimysium reference quality precision, improve the reproducibility of quantitative analysis, and improved signal noise ratio.Other benefit is to have eliminated to carry out low or high laser energy has the ion of broad m/z scope with analysis needs.Single laser current metric density just can be used for surveying all ions low and high mw now, has simplified the analytical method to unknown mixture greatly.

When this device and traditional parallel way of separating when comparing, possible one of them to give the impressive advantage of people be that it does not need ability that the rigidity sample is positioned.Because TOF measures and eliminated from the ion forming process basically, the home position of ion has also just receded into the background.And, because being formed on to need not in the hyperbaric environment to follow and applying the high pressure isolation field and just realized of ion, so the designing requirement of the solid sample that enters system is just greatly reduced.In the good external perimysium reference quality precision property of maintenance, can adopt simple method to replace two-dimentional sample controller.In addition, sample exists the surface no longer to need to make of metal or other conducting medium.

Say that briefly laser desorption ionisation (LDI) Qq-TOF MS has following advantage with respect to existing LDI-TOF MS technology: (1) has improved external perimysium reference quality precision (typically 20-50ppm); (2) improved resolution; (3) improved ms/ms efficient; (4) improved the simplification that produces signal with single high laser energy levels, it has been eliminated needs high and low energy scanning; (5) by using the TDC technology and having improved the quantitative analysis ability than the high 2-4 of minimum desorb threshold value laser current metric density doubly; (6) reduced requirement to two-dimentional sample adjuster; (7) detecting probe surface that sample is existed uses the potentiality (for example, the two-dimentional probe array of injection-molded) of plastic components; (8) by using the single ionic monitoring to reduce chemical noise and having improved and measured the energy of ions that is in EAM chemical noise field.

Laser desorption ionisation (LDI) Qq-TOF MS has following advantage with respect to existing MALDI-PSD method in protein characterizes and discerns.

LDI-QqTOF provides higher material resolution capability and quality precision; In the database mining process, the ability of this raising has reduced the quantity that wrong positive database hits, and has simplified recognition methods.And QqTOF also provides the specific energy sensitivity that enough PSD MS/MS obtain to surpass the sensitivity of an order of magnitude greatly.

Analytical equipment of the present invention has confirmed ability that MS/MS is surprising and the mass distribution error that is lower than 20ppm for single MS analysis.The latter allows a large amount of protein that is retained on the single affinity capture detecting probe surface is discerned simultaneously.

Other parts

Affinity capture probe series connection MS equipment 100 typically also comprises the digital computer that docks with the tandem mass spectrometer detector.Digital computer typically also docks with laser desorption source 12, allows this computer not only to control the generation of ion but also participates in the collection and the analysis of data.

Analysis software can be loaded on computers or be in Long-distance Control, but gets in touch with computer addressablely.For example, the analysis software package that can obtain, for example protein prospector, PROWL or Mascot search engine are used in permission on the World Wide Web (WWW) thereby computer can link to each other with the internet.Analysis software also can reside on LAN or the WAN server out and away.

The affinity capture probe

In order to guide analyte, for example below will describe in detail at our department's branch, at least one affinity capture probe 16 that has adsorbed analyte is engaged on the probe interface 10, and be in can be by the position of laser desorption/ionization source 13 inquiry, and transmits maldi ion and enter tandem mass spectrometer 14.

Probe 16 typically has one or more absorption surface 18, their surface can be different each other (18a, 18b, 18c, 18d).Typically, if a plurality of absorption surfaces 18 are arranged, on all common sides that all is exposed to probe 16.

Absorption surface 18 typically or chromatogram absorption surface or the affine surface of biomolecule.

The affine surface of chromatogram is contained and can chromatogram be differentiated or the adsorbent of separate analytes ability.So this surface comprises anion exchange part, cation exchange part, anti-phase part, metal affinity capture part and mixed mode adsorbent, these terms can be understood in chromatographic technique.The adsorbent that comprises biomolecule that can specific bond is contained on the affine surface of biomolecule.This surface thereby can comprise antibody, acceptor, nucleic acid, lectin, enzyme, biotin, avidin, streptavidin, Staph albumin A and Staph Protein G.Absorption surface is further described part below.

Interface 10 is placed on probe 16 with laser desorption/ionization source 13 to be had on the position of query relation.Typically, wish that laser can inquire about probe absorption surface 18.In addition, interface 10 is placed on probe 16 absorption surfaces 18 position that query relation is arranged with laser desorption/ionization source 13.If 18 of absorption surfaces are positioned on the surface of probe 16, the probe holder at probe 16 and/or interface 10 can form asymmetric size, just like this with direction mandatory be inserted in laser desorption source 13 of probe 16 along absorption surface 18 existence.

When probe 16 has a plurality of absorption surface 18, will wish that lasing light emitter 12 can each absorption surface 18 of direct addressin.This can provide lasing light emitter 12 and/or movable interface 10 by employing, perhaps adopts its combination, its optics is embedded between lasing light emitter 12 and the interface 10 and realizes.

Probe 16 can be the affinity capture probe, as employed in single MS analyzes (for example, those are from Ciphergen biosystem Co., Ltd, Fremont, the commercial acquisition of CA USA) at present.

The application of III affinity probe series connection MS equipment

Above-mentioned analytical equipment of the present invention provides great advantage, and novel method is provided, and is used for (1) protein and finds and discern the interaction that reaches between (2) sign specific bond gametophyte, will describe successively it now.

In general, the advantage of above-mentioned analytical equipment comprises: carry out the ability of high-quality precision measure in one matter MS, and series connection MS pattern and affinity capture probe technique, particularly with special receptors bind system, the ability that combines.

A. the discovery of protein and identification

1. the advantage of the inventive method

A series of relevant issues that protein biology man attempts to solve are discovery, identification and mensuration exploitations (assay development) of protein.The discovery of protein be one a kind of for example because played as the sign of diagnosis or carried out the effect of key cells function and in the interested system of biology angle, find the process of protein.Protein identification is to determine to discern the process of the protein of being found.Measuring exploitation is that exploitation is measured reliably to survey the process of protein.With respect to previous technology, method of the present invention provides advantage for the professional who carries out these processes.

The topmost advantage of the present invention is that it provides can carry out from protein and is found to protein identification to the single platform of measuring the development and operation step.Providing of single platform based on the SELDI technology reduced the time of measuring between confirming from being found to significantly: will spend the operation of time several months when using prior art, and can only need several weeks or a couple of days now.Method of the present invention has also reduced the desired sample size that experimentizes significantly.Needed micromole's quantitative analysis thing in the method formerly, this method can be carried out identical experiment by enough picomole quantitative analysis things.This has just overcome obvious obstacle rare when sample or when being difficult to enrichment.

Before, protein was found to use two-dimentional gel or WesternBlots with separating to be accompanied by.Yet, between gel, be the process of a difficulty relatively mutually with the protein of surveying differential expression.

The protein of being found now can be by using mass spectrometry method identification.Important protein matter can separated and final cracking in the gel of protease is arranged, and peptide fragment can enough mass spectrometers and suitable bioinformatics method analysis.Yet gel and current mass spectrometer method are incompatible, thereby the peptide section must shift out from gel.Because the process of back can cause sample loss inevitably, this method needs a large amount of initial albumen and material.When protein was rare, the situation that may run into as important albumen was just this has increased the difficulty of handling.

In case this protein is identified, the professional just needs exploitation to measure to survey this protein reliably.Typically, it relates to exploitation ELISA mensuration.This technology conversely, needs to produce antibody.This may be a task consuming time, if particularly be difficult to produce under the situation of the quantity that is enough to be used in immunity at protein of interest.

Therefore, previous technology may need three kinds of different technology to realize protein discovery, protein identification and protein determination.Method of the present invention can enough a kind of technology realize these.

2. exploitation (Assay Development) method is found, discerns and measured to protein

The method that is used for discovery, identification and the mensuration exploitation of protein of the present invention comprises that the different mapping (map) of preparation is to find protein or protein of interest matter, with affinity capture probe series connection MS identification of protein with use affinity capture probe laser desorption ionization chromatographic surface to measure or affinity capture probe laser desorption ionization biologic specificity surface measurements is verified.

This process can followingly be carried out.Protein of interest matter is provided or passes through, for example, use retention difference location (difference mapping) research to find protein of interest matter.These methods exist, and for example, among the WO 98/59362 (Hutchens and Yip) explanation are arranged.Briefly, two kinds (for example, normal and ill in some importance; Functional nand function) the retention chromatographic process detection of different biological samples.This method comprises sample is exposed under the affine and wash conditions of a plurality of different chromatograms, then detects " reservation albumen " with affinity capture probe laser desorption ionization.The albumen of differential expression is to make the further material standed for of detection between two samples.Because they detected on mass spectrometer, so the molecular weight of these material standed for albumen is known.

Usually, except protein of interest matter, also have numerous protein to be retained on the chip.Therefore next selection step is to improve affine and wash conditions, simplifies sample thereby protein or protein of interest quality guarantee are stayed to further analyzing.(these also have explanation in the patent disclosure of Hutchens and Yip.Although) more satisfactory to catching of single protein of interest, catch and be no more than about 10 can to detect albumen then more desirable.This method of refining provides improved chromatographic determination for protein of interest.

Keep then albumen on probe with selected proteolysis reagent cracking, be that research subsequently produces the peptide pond.With special integrated protein enzyme for example insulin digestion have more advantage because this cracking pattern is known and and bioinformatics method, comprise being stored in that the silicon of protein dissociates compatibility in the database.Then the product peptide with high-resolution, high-precision MS-MS (for example have the mass distribution error less than 20/1000000ths and resolution about 10,000) analyze.At this moment, seeming product or a certain other that not clear special peptide section is interest albumen keeps the product of albumen.However, this analysis still by select a peptide fragment (may be at random, may based on the corresponding to information of interest albumen) and this peptide carried out vapor-phase thermal cracking and carry out.One of them this type of method is collision induced dissociation (CID).Peptide does not need to separate from the core product, because MS-MS equipment separates the interest peptide in mass spectrometer with other peptide.This will produce the further cracking pattern of selected peptide section.

Use the method set up technically, database digging agreement for example, thus the information that obtains from fragmentation mode is used for the query protein database supposition identification material standed for one or more for the protein that produces this peptide section generates.This agreement is carried out the method that the coupling tightness is analyzed usually, and it weighs the prediction mass spectrum and the actual mass spectral matching degree of selection fragment of protein.Then protein-based in the database in albumen fragment and database albumen consistency confidence level measurement and defined the level.Understanding is the known parent albumen and the quality of original species, will help to limit the identification quantity of the material standed for that is produced.

Then, the supposition characteristic protein matter that produces peptide fragment is checked.Utilization is from supposition feature material standed for elementary sequence library and the knowledge of obtaining in the dissociative pattern of employed proteolysis reagent, just can predict peptide fragment, especially and their molecular weight, this be from the fragment that the feature material standed for is dissociated by proteolysis reagent should produce.Then, these a series of precognition fragments are compared with the actual fragment series that the proteolysis that is retained in based on quality on the chip is produced afterwards.If calculated the precognition fragment, so just can be sure of to suppose that the feature material standed for is consistent with the identification that certain is retained in the albumen on the chip practically.If it is identified up to the protein that produces fragment no, so just must to test other supposition feature material standed for by elimination methods.Under this situation, what produced can exclude from the fragment that had calculated that is produced is all with the corresponding to fragment of Recognition Protein.

If improved affine and elution requirement after have only a kind of protein to remain, so just can calculate whole peptide fragments, thereby just this process be through with.Yet, remaining if having more than a kind of protein, situation just may be complicated more.For example, employed fragment can be produced by protein of interest matter in the analysis, and perhaps it can be by being retained on the chip but not be that the protein of proteins of interest produces.In this case, the step that repeats the analysis of the peptide fragment that do not calculate with the MS-MS method of illustrated mistake is useful up to identifying protein of interest matter or identifying all reservation albumen.

At last, proteins of interest can be by passing through affinity capture probe laser desorption ionization method, perhaps adopt to be retained the chromatographic surface that protein determines or can be developed and be used for the biologic specificity surface of in the quantitative analysis of affinity capture probe laser desorption ionization, using doing quantitative analysis.The foundation of biologic specificity surface is included as Recognition Protein provides binding partners, and such as antibody, perhaps acceptor if a kind of acceptor is known, and is combined in the surface of chip with it.Then, protein of interest matter just can have been done quantitative analysis by enough SELDI that had illustrated.

B. the sign of interaction of molecules

Analytical equipment of the present invention makes for the first time becomes possibility for Study of Interaction between the specific bond gametophyte provides a kind of sensitivity, method effective, single platform.

Specific bond interacts and is in the core of broad-spectrum biological process.In addition, measuring and characterize this interactional ability is essential prerequisite for this process of overall understanding; On clinical level, measure and characterize this interactional ability and can be used in adjusting, perhaps even to abolish this interactional reagent be very important for understanding the not normal and appropriate design of pathology in this process.

Organize on the level at organized eucaryon, for example, the intercellular signal of mammalian nervous system is to propagate by the interaction between neurotransmitter and its homoreceptor.The molecular mechanism of understanding this binding interactions is essential for overall understanding sort signal mechanism.On clinical level, understand the mechanism of the molecular mechanism of this binding interactions for overall understanding signal symptom, with the medicament that alleviates the sort signal symptom for appropriate design be essential, medicament helps treatment of diseases, its scope from Parkinson's to schizophrenia, from the compelling sex behavior disorder to epilepsy.

On cyclical level, for example, the interaction of B-cell receptor and circulating antigen is that the expansion of inducing B cell clone, differentiation and antibody specificity humoral immune reaction are necessary.For the understanding of the antigenic determinant that helps antigen recognizing, be very crucial to the overall understanding immune response.On clinical level, it is important that this understanding provides the vaccine of stronger humoral immunity for design.Similarly, there are interaction between the associated peptide of MHC on the cell in TXi Baoshouti and proof and antigen immunity are crucial for trigger cell.It is important providing the vaccine of stronger humoral immunity to the understanding of the T cell determinant that helps antigen recognizing for design.

On the individual cells level, the phenotypic response of pair cell external signal is by at least one, be a series of intercellular interaction more frequently, in nuclear kytoplasm, interact to transduction signal from the start-up phase mutual effect of cell surface receptor and part, transcribe the interaction of the factor and DNA to protein again, and propagate, caused can observed phenotypic response for the different mode of gene expression then.

For example, estrogen and progesterone are essential by the difference combination of gonad cell for ovulation.Understand steroid hormone receptor and hormone part, and on the other hand, the molecular mechanism of understanding the binding interactions of ligand receptor and genome steroid hormone response element is important for understanding hormone response.Conversely, this understanding is for understanding infertility, and is intended to cancel the medicament of ovulation, implantation and/or fetus viablity for appropriate design---for example RU486---is important.

This interaction is not only in eukaryotic system, and in prokaryotic system and also can find in the interaction of protokaryon and eucaryon.For example, some Gram-negative bacteria has the necessary pili of the eucaryon eurethra of intrusion; Thereby understand this interaction for this pathologic process of overall understanding, and can prevent that for appropriate design the medicament of this intrusion from being important.

There have been many technology to be used to this technical field, have been used for intermolecular interaction between research and location (map) specific bond gametophyte.Each all has significant advantage.

In first method therein, a specific bond gametophyte member is fixed on the adsorbent that is filled in the chromatographic column.For the position in the structure of locating second (freedom) binding partners that (map) and first (combined) binding partners contacts, second (freedom) binding partners is dissociated.Although special chemistry dissociates (for example, passing through CNBr) or even non-special chemical hydrolysis can accomplish that also typically, this dissociating is proteolytic enzyme by special.Therefore, digestive juice by this post with in conjunction with those still with second (freedom) binding partners of first (fixing) binding partner binds.

Then, with salt or pH gradient elution, then peptide is identified by using the guiding of MALDI or electron spray ionisation to enter in the mass spectrometer peptide of second binding partners by typically.

This method has a plurality of well-known, and significant problem.At first, need first binding partners of large-scale purification to produce special absorption.The second, need in a large number, the typical case is a purifying, second binding partners is to be used for digestion, desorb and elution, because each of these stages all exists dilution effect and analyte loss.And although mass spectral analysis subsequently may be highly sensitive, yet liquid phase analysis is connected with MS and also can produces the loss of analyte once in a while.

Perhaps more basic shortcoming is, by second binding partners that dissociates before in conjunction with first binding partners, makes and has only those suitably to keep peptide fragments of its molecular structures just to understand combination on second binding partners, and be detected subsequently.If for example, antibody is discontinuous with combining of antigen, rather than linear, determinant, the cleaved destruction of this discontinuous determinant meeting; Can not keep again and the combining of immobilized antibody, thereby this antigenic determinant just can not be detected.

Second method in this technology is to use point mutation to be positioned at that those help the residue of intermolecular combination in the protein combination gametophyte.

Back one method need be cloned the protein bound gametophyte, produces the point mutation of expectation, the protein expression of recombinant conversion, with and purifying.Then, measure the binding kinetics of other binding partners of transforming protein, thereby determine of the influence of sudden change residue intermolecular interaction.

Except frequent use, the X-radiocrystallgraphy that the contact situation between the binding partners also can enough binding partners illustrates.This technology height is effective, and the resolution of atomic level can be provided, but needs each binding partners all highly purified, but also needs to form suitable crystal.

Affinity capture tandem mass spectrometry equipment of the present invention provides improved method, and its needed parent material is wanted much less, has got rid of point mutation analysis, crystallization, and has reduced the requirement of purifying significantly.

The first step is that one of them of binding partners is fixed on the affinity capture probe.

Each binding partners can both be fixed; Yet be binding partners freely, it is relevant will to be acquired in conjunction with contacting structure information.Utilize the model of the interaction of receptor/ligand, part is fixed on the probe to allow identification receptor to participate in the zone of binding partner as this method; Part is fixed on the zone that also will allow recognition ligand participation bind receptor on the probe.---for example proteohormone, cytokine or chemical activator (chemokine)---uses the separating experiment of each binding partners will produce bilateral understanding to intermolecular contact in turn when part is protein.

The gametophyte of probe combination can be fixed by non-covalent interaction covalency or strong.The availability of appropriate reaction group on this gametophyte that is about to fix and the chemical nature of detecting probe surface are depended in this selection.Suitable chemical property is well-known in analysis technical field.

For example, when being about to immobilised binding partners and having the free amino group acid groups, between the phosphinylidyne diimidazole of the free amino group acid groups of secure bond gametophyte and detecting probe surface, can form covalent bond.Similarly, also can utilize the free amino acid of secure bond gametophyte or thiol group that this binding partners is covalently bound on the detecting probe surface with epoxide group.Strong coordinate bond or coordination valence (dative) bond energy reach between the free sulfhydryl group group of secure bond gametophyte and gold on the detecting probe surface or platinum and form.

Then, selectively, the residual reaction site on the detecting probe surface can be closed to reduce the non-specific binding to the activation probe.

Then, second (freedom) binding partners contacts with the affinity capture chip and is allowed to and first (fixing) binding partner binds.

If second binding partners is known and can obtains, perhaps, more typically, be from heterogeneous mixture, for example suspect and catch out in the biological sample contain second binding partners that second (freedom) binding partners can be temporary transient pure in solution.This biological sample, as in the discover method of the biomarker of describing not long ago, can be biofluid, for example blood, serum, blood plasma, lymph, interstitial fluid, urine, juice, also can be cytolysis thing, emiocytosis thing, the perhaps component of part fractionation and purifying wherein.

Then, this probe washes with the eluent of the definite elution property of having of one or more.These flushings are used to reduce the quantity with the species of the non-specific bond of probe.

Then, the energy absorbing molecules is joined, typically, in the liquid phase, and be allowed to drying.The application of energy absorption molecule can be affected, and its mode with the suffered influence of present employed affinity capture probe is identical; When use protein-chip  array (Ciphergen biosystem Co., Ltd, Fremont, CA, in the time of USA), the energy absorption molecule uses according to the operation instructions of manufacturer.

Then, non-covalent material in conjunction with the affinity capture probe---as with the molecule of second binding partners of first (fixing) binding partners specific bond, the molecule of non-specific bond detecting probe surface, non-specific bond first binding partners---be detected in mutually at laser desorption ionisation mass spectral first.

Mass spectrometer can be a single-stage affinity capture LDI-MS equipment, such as Ciphergen biosystem Co., Ltd (Fremont, CA, PBS II USA).But affinity capture of the present invention series connection MS provides higher quality precision and the mass resolution of Geng Gao, from but preferential the selection.

Typically, second (freedom) binding partners can know from research not long ago, and its existence or do not exist and can promptly determine with mass spectrum.The unknown of second if (freedom) binding partners, then each material that is attached on the probe may be all detected successively.If it is too high to detect the quantity of species, can wash this affinity capture probe with eluant, eluent, to reduce the quantity of the existing species that are used to analyze with different elution property (typically, stringency increases progressively).

In case second the combining of (" freedom ") binding partners and first (fixing) binding partners is identified, second binding partners is just dissociated.This typically is accompanied by second binding partners (in this case, its non-covalent ground, but specifically with first binding partner binds, in turn, first binding partners is fixed on the detecting probe surface) with special integrated protein enzyme, as insulin, Glu-C (V8) protease, integrated protein enzyme Arg-C (or serine protease, or cysteine proteinase Arg-C enzyme), Asn-N protease, perhaps Lys-C protease, contact.

After the digestion, peptide is surveyed by mass spectrum.

If all fragments of second binding partners all will be discerned---for example, be used to confirm by the identification of peptide quality fingerprinting analysis to second binding partners---can apply the energy absorbing molecules and enter mass spectrum by laser desorption ionisation guiding peptide with probe.For this purpose, can use the linear TOF MS of the single accelerating stage of Ciphergen PBS II; The present invention can provide more that the series connection MS of high-quality precision and mass resolution is preferential the selection, because resolution that increases and precision have reduced the quantity that the supposition returned " is hit " from any given data base querying on any given level of confidence.

Yet more typically, still wish to analyze those and immobilization first binding partner binds fragment of second binding partners the most closely.In this case, before adding the energy absorbing molecules, with a kind of or more eluant, eluent flushing probe.

In this case, probe is inserted into the present invention and connects in the middle of the interface of MS, and then the fragment of second binding partners (being typically peptide) is detected.

If the identification of second (freedom) binding partners is known, the quality that then is detected fragment by with resolvase known dissociate rule application in the primary amino acid sequence of second binding partners, just can make comparisons with institute's predicted quality.In this method, each fragment can both be identified, so just determined the position that causes in the structure of second binding partners with the part of first binding partner binds.

Although, can use single-stage MS equipment in theory, in fact also existing is not the fragment that produces from second binding partners, this makes to analyze and becomes chaotic.Therefore final identification meeting generally is benefited from the high-quality resolution rate of present device and quality precision, and further can be benefited from MS/MS analyzes.

The unknown of second if (freedom) binding partners, then this binding partners can be analyzed identification by enough MS/MS.

Typically, following form is adopted in this analysis, promptly selects first parent peptide in the phase I of MS, the peptide of selecting is dissociated, and the second stage of analyzing at MS then produces the mass spectrum of fragment.In gas phase, dissociate and preferably realize by collision induced dissociation.In the preferred embodiment of affinity capture tandem mass spectrometry instrument of the present invention, CID can be subjected in q2 and about 10 ^-2The influence that the nitrogen of holder collides.

Then, utilize this fragmentography, as Yates et al., U.S. Patent number Nos5 by known algorithm, 538,897 and 6,017,693 is disclosed, and protein prospector MS-TAG (http://prospector.ucsf/edu) module is employed, comes the search sequence database.

The identification of supposing can be further be verified by selecting second parent peptide and repeat this method, as for confirm all peptides all come from a kind of certifiable parent needed.

Thereafter, just as mentioned above in case second binding partners is identified, the mechanism of intermolecular interaction just can be studied.With lyases (or chemical reagent, as CNBr) known dissociate rule application on the elementary sequence of second binding partners that has just identified, and the peptide of experiment measuring is navigated in the theoretical digestion (theoretical digest), just identified thus and be combined on immobilization first binding partners, and therefore in natural molecule, also helped the peptide that combines with immobilization first binding partners.And as mentioned above, can repeat this experiment by the wash-out that increases progressively stringency to discern those combinations peptide the most closely.

Also can carry out the mechanism of other perturbation with the intermolecular combination of further explaination.

Can change the elution property of washing the eluant, eluent of probe after the second binding partners cracking, facilitate the fragment of strong interaction with identification, perhaps identification helps the contact that pH relies on or salt relies on of combination.

That yes in chromatogram and technical field of molecular biology is well-known for this principle, that is: along with the rising of wash-out stringency (for example, salinity rises, and temperature raises), those and the more untight fragment of immobilization first binding partner binds will elute from first binding partners.In existing geometric system, thisly will elute from probe, and in mass spectral analysis subsequently, lose in conjunction with relatively poor fragment.Therefore, can carry out a series ofly, the probe of its probe or identical homologue is with the experiment of the stringency that increases progressively flushing, so just produced the subclass of the second binding partners fragment of a series of classifications.

As mentioned above, first (fixing) and second (freedom) binding partners can exchange, thus allow wash-out another one binding partners in conjunction with the contact site.

More useful perturbation (perturbation) is to remove or change back translation modification on one or two binding partners.For example, if first binding partners is a glycoprotein, before in conjunction with second binding partners and/or afterwards, handles with one or more species specific or non-specific glucoproteinases and will help to explain the contribution of saccharide residue combination.

Similarly, when one of them binding partners is nucleic acid, after combining another binding partners, handles the nucleic acid binding partners and can help to discern crucial in conjunction with residue with nuclease.

The method of sign intermolecular interaction recited above has replaced multi-platform, the high labour intensity of prior art, the technology operation of muting sensitivity, replaces single platform, more efficient, highly sensitive method.This method can be applied among the multiple different biosystem and problem.

Advise that as top method of the present invention can be used in the location (mapping) of antigenic determinant---just, help the contact site of binding antibody, TXi Baoshouti or MHC in the identification antigen.This method can be used in bio-ligand and its acceptor in the explaination multiprotein complex, transcription factor and nucleic acid, the mechanism that combines of transcription factor and other transcription factor.

Although albumen/protein-interacting has been discussed in top special discussion, method of the present invention can illustrate between lectin and the glycoprotein with putting into practice, between protein and the nucleic acid, reaches the binding interactions between micromolecule and the acceptor.

Especially for the micromolecule part, this method also can be applied to design the activator and the antagonist of known receptor.

In 10 years, many technology have grown up and have been used for a large amount of micromolecule of combination results in the past, and have various uniformly and living cells screen the ability that these molecules are used to influence the bioprocess of one or more in measuring.For example, evenly scintillation proximity is measured and can be used for screening the combinatorial libraries that combines with known receptor; The compound that can be used in the screening combinatorial libraries based on the digital raji cell assay Raji of image is used for downstream effect, and the cytoplasm/nucleus transhipment as acceptor changes the distribution of intracellular Ca2+, changes cellular activity.

But,, will help to design advisably molecule with better pharmacokinetics and acology index to the interactional understanding of micromolecule and its acceptor in case this lead compound is identified.Technology of the present invention is very suitable for this purposes.

If the signal that micromolecule provides approaches the signal that draws by the energy absorption molecule, MS will carry out with single ion surveillance style so, thereby only seek known substances in the combinatorial libraries component.

Give an example 1

The identification of prostate cancer biomarker

Traditionally, prostate cancer is diagnosed by tissue biopsy after the prostate-specific antibody (PSA) of finding blood levels increases.In normal male, PSA exists level to be lower than 1ng/ml.For BPH and carcinosarcoma of prostate, the PSA level can be brought up to 4-10ng/ml.Chen?et?al.J.Urology?157：2166-2170(1997)；Qian?et?al.，Clin.Chem.43：352-359(1997)。PSA is known to have chymotrypsin activity, can cracking tyrosine and leucic C end.Qian?et?al.，Clin.Chem.43：352-359(1997)。

Use protein-chip  differential display technique to analyze the seminal plasma that obtains from BPH patient and patients with prostate cancer.Fig. 3 has shown the test figure of single BPH and patients with prostate cancer spermatin.Virtual (virtual) gel shows the visual comparison that is used to improve between the sample.The difference site that prostate cancer deducts the protein test figure of BPH is presented at the below of gel observation station.The positive shows signal hint protein in difference site has been raised (upregulated) in prostate cancer, and on behalf of the downward albumen of prostate cancer, negative peak regulate.Detected the last tonal signal of a plurality of uniquenesses, this shows it may is the biomarker of prostate cancer.

The albumen that one of them this quilt of separation raises on probe is by using mixed mode surface and neutral pH buffer solution flushing (see figure 4) to realize.In this case, this albumen by enrichment with near balanced (homogeneity).Then, the biomarker material standed for of this enrichment is digested with original position with insulin exposure.After the cultivation, add saturated CHCA (matrix) solution and analyze digestion product with SELDI-TOF subsequently.

There is multiple peptide to be detected out (see figure 5).The peptide signal that generates is submitted carrying out the Protein Data Bank analysis, and carries out the preliminary identification of human semenogellin I.Some complexity of this identification, because biomarker has the molecular wt of about 5751Da, (MW 52,131Da) much smaller than the weight of semenogellin I.

Identical protein purification is submitted to detect (see figure 6) to be used for protein-chip LDI Qq-TOF MS.Because be in the parent ion of 5751Da, surpassed the quality limitations (3000M/z) that present LDI Qq-TOFMS/MS analyzes, therefore used divalent ion to be used for CIDMS/MS ordering (see figure 7).CID MS/MS result is used to carry out the Protein Data Bank digging.15 kinds of mapped (mapped) the Huis' class seminal fluid basic proteins (SBP) in 26 kinds of MS/MS ions, the proteolysis of semenogellin I is derived fragment, and the final identification of this candidate's biomarker is provided.

Although for example these preliminary research have shown the biomarker of protein fast, the full confirmation of any biomarker all need to be parsed into beat or even associated sample up to a hundred to obtain about expressing and the statistically evident information of the incidence of disease.

All patents of mentioning herein, patent announce and the list of references of other public publication all hereby integral body quote and think reference, each is all independently and particularly with reference to quoting.By quoting of various lists of references herein, the applicant does not admit that any special list of references is its invention " prior art ".

Although concrete example is provided, top description is illustrative, is not limited thereto.Any or more feature of the embodiment that had before illustrated can be by any way combines with one or more the feature of any other embodiment of the present invention.And when checking specification, multiple variant of the present invention will be conspicuous for those technical staff in the art.Therefore, scope of the present invention should not determine according to top description, but should according to additional claims and completely the equivalent scope determine.

Claims (63)

1. an analytical equipment comprises:

The laser desorption ionisation source;

Affinity capture probe interface; With

Tandem mass spectrometer,

Wherein the affinity capture probe can be meshed in this affinity capture probe interface, and with this probe be placed on this lasing light emitter have query relation and simultaneously with the position of this tandem mass spectrometer contact.

2. the analytical equipment in the claim 1, wherein this laser desorption ionisation source comprises laser-excitation source and laser light path system, and this laser light path system can be sent to this probe interface from this laser-excitation source with excitation photon.

3. the analytical equipment in the claim 2, wherein this laser light path system is sent little Jiao's of about 20-1000 energy from this laser-excitation source to every square millimeter of inquiry detecting probe surface.

4, the analytical equipment in the claim 2, wherein this laser-excitation source is selected from the group that comprises continuous wave laser and pulse laser.

5, the analytical equipment in the claim 2, wherein this laser-excitation source is from comprising nitrogen laser, Nd:YAG laser, erbium: YAG laser and CO ₂Select in the group of laser.

6. the analytical equipment in the claim 2, wherein this laser-excitation source is pulse nitrogen laser.

7. the analytical equipment in the claim 3, wherein this laser light path system comprises the optics of selecting from the group that comprises lens, reflective mirror, prism, attenuator and light beam dispenser.

8. the analytical equipment in the claim 3, wherein this laser light path system comprises the optical fiber with input and output, wherein this laser-excitation source and this optical fiber input are coupled.

9. the analytical equipment in the claim 8, wherein this laser light path system also comprises optical attenuator.

10. the analytical equipment in the claim 9, wherein this attenuator is placed between this laser-excitation source and this optical fiber input.

11. the analytical equipment in the claim 9, wherein this attenuator is an optical coupler, and this coupler is coupled in this optical fiber input with this laser-excitation source.

12. the analytical equipment in the claim 9, wherein this attenuator is positioned between this optical fiber output and this probe.

13. the analytical equipment in the claim 8, wherein this optical fiber output has the maximum gauge of about 200-400 μ m.

14. the analytical equipment in the claim 13, wherein this optical fiber input has the diameter of about 400-1200 μ m.

15. the analytical equipment in the claim 2, wherein this laser desorption ionisation source also comprises probe viewing optics device.

16. the analytical equipment in the claim 8 also comprises optical coupler, this coupler is coupled in this optical fiber input with this laser-excitation source.

17. the analytical equipment in the claim 16, wherein this coupler or this fiber is bifurcated and isolate part energy from this laser-excitation source.

18. the analytical equipment in the claim 17, wherein this coupler or this optical fiber are bifurcated and allow to guide visible illumination desorb point.

19. any one analytical equipment in claim 15 or 18 also comprises the CCD camera, the light that this CCD camera is positioned and returns from this probe reflection to survey.

20. the analytical equipment in the claim 1, wherein this affinity capture probe interface comprises probe holder, and this probe holder can reversibly mesh this affinity capture probe.

21. the analytical equipment in the claim 20, wherein this affinity capture probe interface also comprises the probe introducing port, and this probe introducing port can reversibly mesh this probe holder.

22. the analytical equipment in the claim 21, wherein this affinity capture probe interface also comprises probe location governor assembly and interface ion gathering system, when this probe holder is engaged in this interface, this probe location adjuster can contact this probe holder, and movably locatees this probe holder and this probe with respect to this laser-excitation source and this ion collection system.

23. the analytical equipment in the claim 22, wherein this adjuster can translation or this probe holder of positioning of rotating.

24. the analytical equipment in the claim 22, wherein this interface also comprises the vacuum-evacuate system, and this system, coupled is in this probe introducing port.

25. the analytical equipment in the claim 24, wherein this vacuum-evacuate system can produce pressure below atmospheric pressure in this probe interface.

26. the analytical equipment in the claim 1, wherein this tandem mass spectrometer is selected from the group that comprises QqTOFMS, ion trap MS, ion trap TOF MS, TOF-TOF MS and fourier transform ion cyclotron resonance MS.

27. the analytical equipment in the claim 26, wherein this tandem mass spectrometer is QqTOFMS.

28. the analytical equipment in the claim 2, wherein this tandem mass spectrometer is QqTOF MS, and this laser-excitation source is pulse nitrogen laser.

29. the analytical equipment in the claim 1, wherein this tandem mass spectrometer has the external perimysium reference quality precision of 20-50ppm.

30. the analytical equipment in the claim 1, wherein the laser current metric density on this probe approximately be minimum desorb threshold value 2-4 doubly.

31. the analytical equipment in the claim 1 also comprises:

The affinity capture probe, wherein this affinity capture probe is engaged in this affinity capture probe interface and is placed on position and the while and this tandem mass spectrometer contact that query relation is arranged with this lasing light emitter.

32. the analytical equipment in the claim 31, wherein this affinity capture probe has the sample absorption surface that at least one is positioned to have with this lasing light emitter the query relation position.

33. the analytical equipment in the claim 32, wherein this at least one sample absorption surface is selected from the group that comprises chromatogram absorption surface and the affine surface of biomolecule.

34. the analytical equipment in the claim 33, wherein this at least one sample absorption surface is the chromatogram absorption surface.

35. the analytical equipment in the claim 34 is selected in the group on wherein this chromatogram absorption surface, anion exchange anti-phase from comprising, cation exchange, immobilization metal affinity capture and mixed mode surface.

36. the analytical equipment in the claim 33, wherein this at least one sample absorption surface is the affine surface of biomolecule.

37. the analytical equipment in the claim 36, wherein this biomolecule is selected from the group that comprises antibody, acceptor, nucleic acid, lectin, enzyme, biotin, avidin, streptavidin, Staph albumin A and Staph Protein G.

38. the analytical equipment in the claim 31, wherein this affinity capture probe has a plurality of being placed on this lasing light emitter the locational separately addressable sample absorption surface of query relation is arranged.

39. the analytical equipment in the claim 38, wherein each this separately addressable sample absorption surface is from comprising the reverse-phase chromatography absorption surface, the anion-exchange chromatography absorption surface, the cation-exchange chromatography absorption surface, immobilization metal affinity capture chromatogram absorption surface, mixed mode chromatogram absorption surface, the affine surface of antibody, the affine surface of acceptor, the affine surface of nucleic acid, the affine surface of lectin, the affine surface of enzyme, the affine surface of biotin, the affine surface of avidin, the affine surface of streptavidin, select in the group on affine surface of Staph albumin A and the affine surface of Staph Protein G.

40. the analytical equipment in the claim 38, wherein these a plurality of separately addressable sample absorption surfaces comprise at least two kinds of different absorption surfaces.

41. the analytical equipment in the claim 1 also comprises:

Digital computer,

Wherein this digital computer is connected with the detector of this tandem mass spectrometer.

42. the analytical equipment in the claim 41 also comprises software program, this software program is carried out by this digital computer.

43. the analytical equipment in the claim 42, wherein this software program this locality is positioned this computer.

44. the analytical equipment in the claim 42, but wherein this software program right and wrong this locality but this computer of communications access.

45. the analytical equipment in the claim 42, wherein this software program can be controlled this laser desorption ionisation source.

46. the analytical equipment in the claim 42, wherein this software program can be by at least one aspect of this tandem mass spectrometer control data collection.

47. the analytical equipment in the claim 42, wherein this software program can be carried out at least one routine analyzer on the data that obtain by this tandem mass spectrometer.

48. the analytical equipment in the claim 42, wherein this software program can be controlled this laser desorption ionisation source, at least one aspect that this tandem mass spectrometer control data is gathered can be passed through, and at least one routine analyzer can be on the data that obtain by this tandem mass spectrometer, carried out.

49. one kind is used to analyze at least one method that detects albumen and comprises:

(a) on the affinity capture protein-chip, catch one or more test proteins;

(b) use proteolysis reagent on protein-chip, to produce the protein cleavage product of test proteins; With

(c) analyze at least one protein cleavage product with tandem mass spectrometer, wherein analysis comprises:

(i) the protein cleavage product separated from protein-chip be drawn onto the gas phase producing corresponding parent ion peptide,

(ii) select the parent ion peptide to be used for subsequently size degradation with first mass spectrometer,

(iii) parent ion peptide that size degradation should be selected under the selected size degradation condition in gas phase with produce the product ion fragment and

(iv) produce the mass spectrum of product ion fragment;

Thus, this mass spectrum provides the analysis to test proteins.

50. the method in the claim 49 also comprises:

(d) by submitting mass spectrum to Protein Data Bank digging agreement, determine at least one albumen identification material standed for for test proteins, this Protein Data Bank digging agreement is discerned at least one protein identification material standed for for test proteins according to mating the measurement of tightness between the theoretical mass spectrum of protein in this mass spectrum and the database in database.

51. the method in the claim 50, wherein (d) also comprises the quality of test proteins and the original species of test proteins is submitted to agreement.

52. the method in the claim 50 also comprises:

(e) relatively discern material standed for and test proteins, this passes through:

(i) mass spectrum of protein cleavage product in the generation (b),

(ii) the mass spectrum with the protein cleavage product is submitted to computer protocol, this agreement is determined the coupling tightness measurement between the mass spectrum of theoretical mass spectrum and protein cleavage product by the identification material standed for pyrolysis product that uses the prediction that proteolysis reagent produces, thus, this measurement has been indicated corresponding to the protein cleavage product on the protein-chip of test proteins.

53. the method in the claim 52 also comprises:

(f) repeat (c), wherein selected parent ion peptide is not with corresponding by the protein cleavage product of identification material standed for prediction; With

(g) repeat (d) for the selected parent ion peptide in (f).

54. the method in the claim 49, wherein test proteins is the protein of differential expression between first and second biological samples.

55. the method in the claim 54, wherein first and second biological samples derive from normal source and pathology source.

56. the method for a detecting analytes, this method comprises:

The affinity capture probe is engaged in the affinity capture probe interface of claim 1 analytical equipment, this affinity capture probe has in conjunction with the analyte on it;

With this lasing light emitter from this probe desorb and this analyte of ionization or its fragment; And then

Measure this analyte of detection by the tandem mass spectrometer on this maldi ion.

57. the method in the claim 56 also is included in this desorb and ionization steps and reaches before this detection steps afterwards, carries out the step of the collision induced dissociation of this maldi ion.

58. the method in the claim 57 also comprises, after desorb and ionization steps and before the collision induced dissociation step of this maldi ion of execution, selection will be collided the step of a subclass of the ion that dissociates.

59. the method in the claim 58 also is included in this affinity capture probe interface before this affinity capture probe of engagement, and this analyte is adsorbed onto step on this probe.

60. the method in the claim 59, also be included in this this analyte is adsorbed onto on this probe after and be engaged to this probe this probe interface in before, the step that makes this probe and this analyte adhere and contact with the energy absorbing molecules.

61. an affinity capture probe interface is used to mesh the affinity capture probe and this probe is positioned to lasing light emitter query relation and while and tandem mass spectrometer contact are arranged, and comprising:

The affinity capture probe holder;

Affinity capture probe introducing port;

Affinity capture probe location adjuster and assembly;

Vacuum and Pneumatic assembly; With

The interface ion gathering system,

Wherein this affinity capture probe holder can be by this introducing port engagement, this probe holder wherein, in the time of in being engaged on this mouth, be placed with this affine adjuster and assembly and contact, wherein this vacuum and Pneumatic assembly can reduce this probe pressure on every side when meshing with this mouthful, and wherein this adjuster can be located this probe holder to be used for ion collection by this ion collection system.

62. the analytical equipment in the claim 22, wherein this ion collection system comprises electrostatic ionic collection assembly, aerodynamic ion collection assembly and the ion guiding device of selecting from the group that comprises electrostatic ionic guiding device and RF ion guiding device.

63. the analytical equipment in the claim 24, wherein this introducing port evacuation system comprises vacuum pump, pressure sensor, vacuum compatible conduit and connection fittings and the compatible valve of vacuum.

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