CN100483124C - Plate for mass spectrometry, process for preparing the same and use thereof - Google Patents

Abstract

The present invention provides a plate for mass_ spectrometry comprising a support and a PVDF-containing coating (that is, a PVDF-deposited thin layer) thereon, and a method of preparing a plate for mass spectrometry, which comprises coating the support surface with PVDF. The present invention also provides a method of analyte identification comprising subjecting an analyte-containing sample to gel electrophoresis to separate the analyte, blotting the separated analyte from the gel to the above-described plate for mass spectrometry, and subjecting the plate to mass spectrometry, whereby the transferred analyte is analyzed.

Description

Plate for mass spectrometry and its production and use

Technical field

The present invention relates to plate for mass spectrometry (plate for mass spectrometry) and its production and use, described plate can carry out protein group (proteome) analysis in a large number, fast, in high sensitivity.

Background technology

Because the progress of medicine innovation fundamental research, comprise the progress of the fundamental research of molecular biology and genomics (genome science), in these several years, marked change has taken place in the situation of medicine innovation, and is also developing the medicine innovation new method of genomic drug innovation representative.

Yet, still can not be from the clear and definite nucleotide sequences of genome predicted protein matter function (physiological action); Be the technology of connect hereditary information and new drug, need to exist back genome (post-genome).Therefore, protein science (protein analysis) just receives publicity, and its purpose is to separate and differentiate all protein of translating in the above-mentioned nucleotide sequences (report is more than 100000 kinds), and specifies their function.

Although, protein group is meant all protein that constitutes single biosome narrowly, but broadly be meant the organism part of histology or anatomy appointment sometimes, as, all protein that contains in all protein that contains in all protein in the cell liquid of specific cells, the serum, the particular organization etc.In this manual, this term uses with broad sense, but it is evident that the complete set zoarium of sensu lato protein group is sense stricto protein group.

In Proteomic analysis, generally use conventional method, but following problems does not solve still in conjunction with two dimensional electrophoresis and mass spectrophotometry.That is to say that in conventional method, before after the migration sample being carried out mass spectrophotometry, migration gel (migration gel) must split into fragment, must extract protein from each fragment with particular solution.Because the time of cost is long and the requirement of the rapid complex operations of multistep, Measuring Time shortening, plant bulk reduce, the screening of macromethod thing and the robotization of whole device have become extremely difficult.

In addition, there is another problem, promptly is called the problem of " low abundance proteins ".For example, in yeast, have only 100 genes produce yeast gross protein weight 50%.This means that remaining 50% protein is the product of thousands of genes.It is most important protein to biosome in a large number that a large amount of low abundance proteinses contain, and for example regulates albumen and signal transmission albumen, comprises acceptor.But the situation that conventional method produces is: can not reclaim a large amount of and trace amount of protein sample that electrophoresis separates.

In for the trial that overcomes these defectives in electrophoresis and the mass spectral combination technique, and, carrying out various effort in order to illustrate protein-protein interactions.For example, isotope-coded affinity tag method (ICAT:isotope-coded affinity tag; Nat.Biotech., 17:994-999 (1999)), the two-hybrid system in the yeast, BIA-MS-MS, protein array method (protein arraymethod) (solution, chip; Trends Biotechnol., 19:s34-39 (2001)) and the peptidomics that utilizes LC-MS-MS etc. be operable.Especially, as the example of protein array method, can mention solid phase protein array method (chip method; Curr.Opin.Biotechnol., 12:65-69 (2001)), information is combined in liquid phase array method (the fluorescence decoding bead in the nano particle; Clin.Chem., 43:1749-1756 (1997); Nat.Biotech., 19:631-635 (2001); Bar code shape nano particle (barcoded nanoparticles); Trends Biotechnol. (2001), the same) etc.

Simultaneously, the present inventor has proposed a kind of method of Proteomic analysis, its make it possible to simultaneously analyzing film protein and by organize into groups (grouping) memebrane protein can with the interactional compound of its generation (WO 02/56026).

The improvement of measuring method (system), also reported the improved effort in the proteomic image field, but their purpose is not for Proteomic analysis except this, perhaps they technically are difficult to be applied to Proteomic analysis.Reported following method, described method comprises: the Western blotting (blotting) that will be separated by electrophoresis is to polyvinylidene fluoride (PVDF) film, and the band (Electrophoresis of use double spread, 17:954-961 (1996)), framework (Anal.Chem., 69:2888-2892 (1997)) or grease (Anal.Chem., 71:4800-4807 (1997); Anal.Chem., 71:4981-4988 (1999); WO 00/45168) measure being fixed to the film that is used on the mass spectral corrosion resistant plate of MALDI type.These methods are imperfect, reason is that not only these methods are required great effort owing in measuring process pvdf membrane being fixed to plate for mass spectrometry, and the background height, and peak intensity is extremely low relatively, therefore they are not suitable for Proteomic analysis, and the demanding detection sensitivity of Proteomic analysis.

In addition, also reported following method, comprise the direct trace of gel after the electrophoresis to the plate for mass spectrometry of matrix coating and the method for carrying out mass spectrophotometry, and comprise the protein electroblotting to the pvdf membrane that is used for trace, then with the plate for mass spectrometry (US 5595636) of this protein diffusion trace to the matrix coating.Yet, in these methods, when using electroblotting, the problem of existence be in stromatolysis during the trace in the trace damping fluid, and measure them and become and hardly may.On the other hand, when using the diffusion trace, trace efficient is low, so that detection sensitivity is not enough, particularly to the low abundance proteins trace is not enough to the detection sensitivity on the plate for mass spectrometry.

And, reported also that based on the improved method of said method this method comprises uses (apply) in plate for mass spectrometry (GB2312782A) with the nitrocellulose (membrane component that is used for the trace purposes) and the potpourri of matrix.Yet, under the situation of electroblotting or diffusion trace, even this method can not be said fully to solve these problems.

As for plate for mass spectrometry, commonly used aluminium sheet or corrosion resistant plate, and their improvement type for example are coated with the plate for mass spectrometry of monox or are added with the plate for mass spectrometry of hydrophobic group, can commercially buy (Ciphergen makes, and WO 94/28418).Yet, even these technology and product can not always satisfy in a large number, fast and carry out the research and development needs of Proteomic analysis in high sensitivity.

The objective of the invention is to introduce the new method of upgrading than based on the protein group conventional method of analysis of electrophoresis and mass spectrophotometry combination.Particularly, the purpose of this invention is to provide can be fast and carry out the technology of a large amount of Proteomic analysis in high sensitivity.

Summary of the invention

The present invention is based on following discovery: compare with the plate that PVDF is fixed on the membranaceous substrate, provide significantly improved mass spectrophotometry detection sensitivity and accuracy by on substrate, form the prepared plate for mass spectrometry of PVDF thin layer with the PVDF coated substrates.In addition, because this plate for mass spectrometry does not contain matrix when trace, even extra advantage is in electroblotting, matrix is not dissolved yet.

Therefore, the present invention relates to:

1) plate for mass spectrometry, it comprises supporter and the coating that contains PVDF on it;

2) prepare the method for plate for mass spectrometry, it comprises with PVDF and is coated with supporter; And

3) method of discriminatory analysis thing, it comprises that the sample to containing analyte carries out gel electrophoresis, with separate analytes, with isolated analyte trace to above-mentioned 1) in the plate or above-mentioned 2 used of stratographic analysis) method in the plate used of the stratographic analysis for preparing, and the analyte of trace carried out mass spectrophotometry.

By following detailed description of the present invention, other purpose of the present invention and characteristics, and effect of the present invention will be apparent.

Description of drawings

Fig. 1 shows the degree from migration gel electroblotting to plate for mass spectrometry.A represents to move gel (before the trace), and B represents to move gel (after the trace), and C represents plate for mass spectrometry of the present invention (after the trace).The numeric character in left side shows the molecular weight of the protein of corresponding band.This shows: in some protein, because electroblotting, almost whole protein partly migrate to the plate that stratographic analysis is used in the gel.

Fig. 2 shows the amount of laser light (Fig. 2 B) that migration gel and stratographic analysis are accepted with the protein that moves on relation of the position between the plate (Fig. 2 A) and the plate.In this two figure, 1,2 and 3 represent stratographic analysis plate, migration gel and protein respectively.In addition, A, B and C represent highlight flux (lightquantity), minimum luminous flux and zero luminous flux respectively.

Fig. 3 shows the relative intensity of the mass spectra peak that use plate for mass spectrometry of the present invention (Fig. 3 A), aluminium sheet (Fig. 3 B) and protein-chip array (H4, Fig. 3 C) are obtained.

Fig. 4 shows use SCUPA that plate for mass spectrometry or aluminium sheet of the present invention obtained and the mass spectrum spectrum (peak image) of HSA.Transverse axis is represented molecular weight, and the longitudinal axis is represented relative intensity.Fig. 4 A and B show the result of SCUPA, and Fig. 4 C and D show the result of HSA.In addition, Fig. 4 A and C have shown the situation of using plate for mass spectrometry of the present invention, and Fig. 4 B and D have shown the situation of using aluminium sheet.

Fig. 5 shown by mass spectrophotometry, uses plate for mass spectrometry of the present invention to carry out the protein (SCUPA) of electroblotting and analysis result of the protein-protein interaction between the antibody (anti-SCUPA antibody) of specific reaction is taken place for it.Transverse axis is represented molecular weight, and the longitudinal axis is represented relative intensity.Numerical symbol among the figure is represented the molecular weight at peak.Interaction between the SCUPA of Fig. 5 A display separation and trace and the anti-SCUPA antibody.In addition, Fig. 5 B shows the result who uses anti-SCUPA antibody to be obtained separately.

Fig. 6 shows the mass spectrum spectrum that is obtained during to pvdf membrane when with the Western blotting of electrophoresis-separation.Transverse axis is represented molecular weight, and the longitudinal axis is represented relative intensity.Numerical symbol among the figure is represented the molecular weight at peak.Fig. 6 A represents the result of SCUPA, and Fig. 6 B represents the result of HSA.

Embodiment

Plate for mass spectrometry

Plate for mass spectrometry of the present invention has coating (thin layer) on supporter (support) (basic structure), described coating contains polyvinylidene fluoride (PVDF).To the material of supporter without limits, as long as it is be used for plate for mass spectrometry one of commonly used.For example, can mention insulator (glass, pottery, plastics/resin etc.), metal (aluminium, stainless steel etc.), conducting polymer, and their combination etc.The preferred aluminium sheet that uses.

The shaped design of plate for mass spectrometry of the present invention is for being particularly suitable for employed mass spectrometric sample inlet.For example, can mention assembly type (cluster type) plate for mass spectrometry etc., described assembly type plate for mass spectrometry was slotted in advance, so that separate independent segment easily, thereby after Western blotting extremely is fit to assembly type (assembly type) plate for mass spectrometry of gel size behind the electrophoresis, each plate is fit to the sample inlet of quality analysis, yet the present invention should not be construed as and is confined to this.

In the present invention, " coating that contains PVDF " is meant the thin layer that forms by with dispersion PVDF molecule deposition on supporter, rather than by the structure (as using conventional known pvdf membrane) that overlapping molding in advance on supporter is crossed prepare the layer.Although preferably use to the mode of PVDF deposition without limits, the method described in the example of the following method for preparing plate for mass spectrometry.

Suitably select the thickness of thin layer, as long as it does not measure generation adverse influences such as sensitivity to the efficient and the mass spectrophotometry of Western blotting, for example, this thickness is about 0.1 to about 1000 microns, is preferably about 1 to about 300 microns.

The method for preparing plate for mass spectrometry

Plate for mass spectrometry of the present invention prepares by PVDF coating supporting body surface.As for the preferred embodiment of coating process, can mention and smear (painting), injection (spraying), vapour deposition, dipping, printing and sputter etc.

Under the situation of " smearing ", (for example can use proper implements, brush), (for example will be dissolved in suitable solvent, as organic solvents such as dimethyl formamides) in suitable concn (for example, about 1 to about 100mg/mL) PVDF (being designated hereinafter simply as " solution that contains PVDF ") smear on the supporter (basic structure, substrate).

Under the situation of " injection ", the solution that contains PVDF with above-mentioned same way as preparation can be ejected from thrower, thereby PVDF is deposited on the supporter equably.

Under the situation of " vapour deposition ", the PVDF thin layer can be formed on the supporting body surface, and it is to form at the vacuum chamber heating and the evaporation PVDF (solid-state or solution) that contain supporter by the vacuum vapor deposition device commonly used that use is used for preparing organic film.

Under the situation of " dipping ", supporter can be immersed in the solution that contains PVDF with above-mentioned same way as preparation.

Under the situation of " printing ", can suitably select and utilize the various printing technologies of using always according to the material of supporter; For example, can preferably use serigraphy etc.

Under the situation of " sputter ", can form thin layer, for example, by between supporter and PVDF, applying high direct voltage, simultaneously inert gas (for example, argon gas etc.) is imported in the vacuum chamber, and make ionized gas collision PVDF, make the PVDF molecule deposition of sputter on supporter.

Can on each surface of supporter, use coating, also can only carry out using coating on the surface of mass spectrophotometry.

Can suitably use the PVDF of preferred form according to coating method; For example, can PVDF be applied on the supporter with forms such as the solution that contains PVDF, the steam that contains PVDF and PVDF solid molecules.Preferably use with the form of the solution that contains PVDF." using " is to instigate PVDF to contact with supporter, so that it is keeping after contact and is accumulating on the supporter.To the amount used without limits; As for the example of the amount of PVDF, can mention about 1 to 100 μ g/cm ²After using, remove by automatic drying and vacuum drying etc. and to desolvate.

Supporter in the plate for mass spectrometry of the present invention (basic structure, substrate) can have the surface, and this surface is before being coated with PVDF, by suitable physics or chemical technology modification in advance (processing).Particularly, can mention polishing surface plate, abrasion (abrasion), acid treatment, alkali treatment and glass treatment (tetramethoxy-silicane etc.) as an example.

Plate for mass spectrometry of the present invention is excellent on stability.That is to say, plate for mass spectrometry of the present invention is characterised in that: though be immersed in the pH value for 2-10 or contain in various salt, the aqueous solution as methyl alcohol or acetonitrile equal solvent or their mixed solvent, under the condition of electric load, moistening and dry repetitive cycling and high vacuum state, adhesive surface (adhesive surface) does not peel off yet.

The purposes of plate

A. differentiate (identification) protein etc.

Use plate for mass spectrometry of the present invention, can differentiate all cpds, comprise protein.That is to say, to (for example containing analyte, protein, nucleic acid, oligonucleotides, sugar, oligosaccharides, cell-membrane receptor activator or antagonist, toxin, virus epitopes (virus epitope), hormone, peptide, enzyme, zymolyte or enzyme inhibitor, co-factor, medicine (drug), agglutinin, antibody etc.) sample (for example carries out electrophoresis, polyacrylamide gel electrophoresis), to isolate analyte, with isolated analyte trace to plate for mass spectrometry of the present invention, and utilize the analyte of mass spectrophotometry trace, so discriminatory analysis thing (on the information of relevant analyte molecule amount).

The sample that contains analyte

The sample that contains analyte can use any technique known to prepare from (biology) material.Herein, example as for (biology) material, can mention: as the cell or tissue of optional biosomes such as animal, plant and microorganism, perhaps liquid in extracellular fluid (for example, blood, blood plasma, urine, bone marrow fluid, ascites etc.), intracellular fluid, the cell granule etc.In addition, also comprise cell culture fluid and the nutrient solution that obtains by genetic recombination etc.

The sample that contains analyte for electrophoretic analysis is carried out in preparation uses known method.For example, under the situation that contains the protein sample, by in the presence of protease inhibitors, in suitable damping fluid after the homogenize target cell, perhaps after using, perhaps after by Hyposmolality shock (shock) smudge cells, perhaps after the ultrasonication cell membrane as cell breaking plant suspension cells such as Polytron, collect centrifuged supernatant, can obtain the part of solubility.In addition, can after utilizing surfactant or protein denaturization agent solubilising, use sediment (soluble) part that is obtained.

Electrophoresis

This is that analyte in the sample goes out the step of (launching) by electrophoretic separation on gel.Employed electrophoretic apparatus can be known device.Can use commercial product.According to purpose, can use any one dimension gel electrophoresis and two-dimensional gel electrophoresis.In two-dimensional gel electrophoresis, the one dimension migration is based on the separation owing to the analyte isoelectric point, and two dimensional migration is based on because the separation of the molecular weight of analyte.To the gel size that is used for electrophoresis without limits.Although 10cm * 10cm is the size of using always, also can use 20cm * 20cm or other size in case of necessity.In addition, although the material of gel mainly is a polyacrylamide, depend on purposes, use other medium, for example Ago-Gel and cellulose acetate membrane also are feasible.As for the concentration of gel, both can use uniform concentration, also can use gradient concentration.

Trace

This is with by the step of the isolated analyte of gel electrophoresis transfer printing from gel (transfer) to plate for mass spectrometry of the present invention.As for blotter, can use known device.Can use commercial product.The trace method itself is known.By the whole bag of tricks (for example, diffusion, electric power and other method), the migration back is transferred on the plate for mass spectrometry at the analyte that launches on the gel.This step is commonly referred to trace.Can mention diffusion trace, electroblotting etc.Be preferably electroblotting especially.

Damping fluid as for using when the electroblotting preferably uses the damping fluid of pH as 7-9 and low salt concn.Particularly, can mention TRIS buffer (Tris buffer), phosphate buffer, borate buffer solution, acetate buffer etc. as an example.As for TRIS buffer, can mention Tris/ glycocoll/methyl alcohol damping fluid, SDS-Tris-Tricine damping fluid etc.; As for phosphate buffer, can mention ACN/NaCl/ isotonic phosphate buffer liquid, sodium phosphate/CAN etc.; As for borate buffer solution, can mention sodium borate/hcl buffer, Tris-borate/edta buffer liquid and borate/ACN etc.; As for acetate buffer, can mention Tris-acetate/EDTA etc.Preferably, damping fluid is Tris/ glycocoll/methyl alcohol damping fluid or sodium borate/hcl buffer.As the composition example of Tris/ glycocoll/methyl alcohol damping fluid, can mention about 10-15 mM (mM) Tris, 70-120 mM glycocoll and 7-13% methyl alcohol.As the composition example of sodium borate-hcl buffer, can mention about 5-20 mM sodium borate.

In addition, behind trace, can add the reagent that is called matrix, absorbing laser, and shift the ionization that promotes analyte molecule, thereby carry out mass spectrophotometry (by the MALDI method) subsequently by energy.As for matrix, can use known matrix in the mass spectrophotometry field.For example, (SPA (=3 for sinapic acid, 5-dimethoxy-4 '-hydroxycinnamic acid (cinammic acid))), indole acrylic acid (IAA), 2,5-dihydroxy-benzoic acid (DHB), alpha-cyano-4-hydroxycinnamic acid (CHCA) etc., however matrix should not be construed as and is confined to this.Preferably, matrix is DHB or CHCA.Under the situation of CHCA, the preferred matrix that is at least 21% saturation concentration of adding.Particularly, can mention the matrix of 21-100% saturation concentration, be preferably the matrix of 40-100% saturation concentration, be preferably the matrix of 50-100% saturation concentration especially.

Mass spectrophotometry

This is by mass spectrophotometry, analyze trace to the plate for mass spectrometry of the present invention analyte and the step of discriminatory analysis thing (from the information of relevant molecular weight).Mass spectrometer is by the ionization gaseous specimen, thereby the molecule of material or molecular fragment enter electromagnetic field, based on the transfer printing state (transfer status) of material, by mass number/charge number separate substance, and the spectrum of mensuration material, and the device of the molecular weight of measurement and detection material.Can use mass spectrometer based on following method principle, described method is the MALDI-TOFMS method, its (matrix-aided laserdeionization that is substance assistant laser desorpted ionized, MALDI) with flight time mass spectrum (time-of-flight mass spectrometry, TOFMS) combination, described MALDI comprises mixing and dry sample and absorbs laser host to produce crystallization, shift by energy from matrix, make crystal current from, and by the instantaneous heating of laser radiation, and the analyte of ionization introduced in the vacuum, described TOFMS comprises that by because initial acceleration, sample molecule ion flight temporal differences is analyzed mass number; Comprise independent sample is placed a drop method of direct ionization analyte from liquid; Nanoscale electrojet mass spectrum (nano-ESMS) method, it comprises the sample solution electrojet to atmosphere, independent analyte is gasificated into the not multivalent ion of folded state; Or the like.

To being placed on the method itself that analyte on the plate for mass spectrometry of the present invention carries out mass spectrophotometry is known.

In addition, be abundant robotization proteome analysis according to the present invention, by moving laser exit towards one dimension direction or two-dimensional directional, perhaps move the pedestal that plate for mass spectrometry is arranged on it on the contrary, and (in the scan method of being interrupted, the existence remaining analyte of laser radiation that is to say to carry out continuous full surface scan, the remaining analyte of Fen Xiing not), can analyze by the electrophoresis one dimension and launch or whole analytes of two-dimensional development.Scan method in conjunction with this method and interruption, make and 96 kinds of samples that are dispensed to 96 orifice plates can be done as a whole embedding (mount) (96 kinds sample on the rectangle chip with constant being spaced) to plate for mass spectrometry, and this sample is carried out mass spectrophotometry.

B. discriminatory analysis thing group

According to the present invention, can analyze the set (analyte group) of multiple analytes simultaneously.That is to say that the sample that contains analyte for preparing from above-mentioned various biomaterials contains multiple and various analyte usually.By separate this analyte group with one dimension or two-dimensional gel electrophoresis, and the analyte group trace that will isolate and be deployed on the gel exists, (be preferably the assembly type plate for mass spectrometry as size and migration gel, it has preformed perforation line, to allow to separating each sheet easily, so that it is meeting mass spectrometric sample inlet behind trace) on the plate for mass spectrometry of the present invention that is consistent, usually can will contain all analytes of containing in the analyte sample (if analyte is a protein, then be protein group), be separated to corresponding position, and with their traces to plate for mass spectrometry.By using above-mentioned continuous sweep type mass spectrometer, measure these analyte groups, can differentiate the analyte group of trace simultaneously.

C. discriminatory analysis thing compound

According to the present invention, analysis of analytes and this analyte is had keyed jointing between the analyte of affinity (and it interacts) simultaneously.That is to say, by utilizing electrophoretic separation of analytes, with isolated analyte trace to plate for mass spectrometry of the present invention, add then contain to this analyte have affinity (interacting) with it compound sample (for example, peptide, protein, nucleic acid, non-peptide compound, synthetic compound, fermented product, cell extract, plant extracts, animal tissue's extract, cell membrane fraction (fraction), organelle film level is graded), to form compound onboard, and the compound that forms carried out mass spectrophotometry, can differentiate the analyte of trace, this analyte had the compound of affinity (interacting with it) and/or their compound (from the information of relevant molecular weight).

For describing the present invention in more detail, below provide embodiment; Yet these embodiment should not be construed as restriction the present invention.

Embodiment 1: the preparation plate for mass spectrometry

Aluminium base structure (aluminium sheet) immersed 1% tetramethoxy-silicane/1% acetic acid solution, and at air drying, calcining is (130 ℃ then, 3 hours), described aluminium base structure is prepared into the inlet of the plate for mass spectrometry that meets protein-chip system (ProteinChip System) (being made by Ciphergen).To be dissolved in the polyvinylidene fluoride (PVDF) in the dimethyl formamide (DMF), be applied on this plate with the amount of 10mg/mL.The plate for mass spectrometry of preparation is flat, and it is of a size of 78 millimeters * 8 millimeters * 2 millimeters, and it has white surface.

In later step, chemical change, electricity variation, machinery variation or physical change take place in plate for mass spectrometry anything but, for example break, peel off, damage and fade, described later step, for example be immersed in the organic solvent with pre-service, contact with running gel, in various damping fluids electroblotting, and high vacuum and drying and mass spectrophotometry during matrix is subsequently used.

Use this plate for mass spectrometry, carry out following research.

Embodiment 2: with the protein electroblotting to plate for mass spectrometry

After (Bio-Rad manufacturing) carries out the 12%SDS-polyacrylamide gel electrophoresis to prestained molecular weight marker, under the 90mA electric current, on the plate for mass spectrometry with the preparation to the embodiment 1 of the protein electroblotting on the gel in 12.5mM Tris/96mM glycocoll/10% methyl alcohol damping fluid.Therefore, although the small number of molecules amount is to remain in (Figure 1B) in the polyacrylamide gel after the prestained Western blotting of 90000-110000, all proteins is by trace (Fig. 1 C) to plate for mass spectrometry of the present invention effectively.

Embodiment 3: the mass spectrophotometry trace is to the protein of plate for mass spectrometry

After sample (protein mixture) is carried out the 12%SDS-polyacrylamide gel electrophoresis, gel is directly contacted with the plate for mass spectrometry of embodiment 1, on the plate for mass spectrometry of the protein electroblotting that will come out by electrophoretic separation preparation to the embodiment 1, and immediately this plate for mass spectrometry is placed in the mass spectrometer and measures.Use 2,5-dihydroxy-benzoic acid (DHB; The 75mg/mL ethanolic solution) as matrix, and use protein-chip system (above-mentioned) as measurement mechanism, the condition of measuring is as follows: detector voltage 1800V, detector sensitivity 8, and laser instrument intensity 280.For mass calibration, utilize myoglobins (available from horse muscle), GAPDH (available from rabbit) and albumin (available from cow's serum), carry out external calibration.In this test, two kinds of protein, i.e. Single-chain Urokinase-type Plasminogen Activator (SCUPA) and human serum albumins (HSA), as protein mixture, under reducing condition, to 4 μ g protein mixtures series connection electrophoresis twice, to the 12%SDS-polyacrylamide gel.That is to say, add initial sample, carried out electrophoresis 40 minutes under the 30mA electric current, turn-off current is added into identical sample in the identical swimming lane once more then, further electrophoresis 23 minutes under the 30mA electric current.After the migration, cut the gel of each migrating channels, further, then the upper surface of two gels with them contacted separately and place, be in contact with one another with plate for mass spectrometry thereafter (Fig. 2 A) in the cutting of the point of addition 39mm place on the gel upper surface.At this moment, with SCUPA, HSA, SCUPA, HSA, HSA, SCUPA, HSA and SCUPA be disposed in order the migration sample, with 4 bands of each protein, 8 band traces are to plate for mass spectrometry altogether.

Because employed mass spectrometer is not continuously, but at 24 equally spaced some irradiating lasers to the single plate for mass spectrometry, so taken place the situation that laser does not expose, part is exposed and exposed entirely, this depends on the position (Fig. 2 B) of the protein of trace on the plate for mass spectrometry.In order to increase the probability to the laser maximum exposure, and therefore be increased in this analytical equipment, the top intensity under this restriction has designed following pattern, promptly by above-mentioned technology, with each model protein trace four different positions to the plate for mass spectrometry.Therefore, even trace protein identical in quality in theory is variable (Fig. 3) available from the peak height of mass spectrophotometry.Therefore, in this experiment, suppose that the value at the peak of maximum relative intensity in a plurality of peaks that obtains is the most approaching with the actual value that laser is exposed fully, discuss.

In advance plate for mass spectrometry is immersed in the methyl alcohol, and (10mM sodium borate buffer liquid, pH8.0) balance were carried out electroblotting 2 hours to it then under the 90mA electric current with the trace damping fluid.Subsequently, carry out mass spectrophotometry, and measure relative peak intensity.For with reference to contrasting, carry out other experiment in an identical manner, difference is to use the aluminium sheet of conventional non-PVDF coating and introduces the aluminium sheet that cetyl is arranged that (ProteinChip array, Ciphergen prepares, H4).The results are shown among Fig. 3 and Fig. 4.

Under the situation of SCUPA, the relative peak intensity of plate for mass spectrometry of the present invention (Fig. 3 A) is 12.31, is 14 times of conventional aluminium sheet intensity level (Fig. 3 B) (0.87).HSA is similarly compared, and the relative peak intensity of this plate for mass spectrometry is 49 times (6.26:0.129) of aluminium sheet intensity level.In addition, the relative peak intensity of plate for mass spectrometry generation is 2 times to 4 times (Fig. 3 C) that H4 produces.And plate for mass spectrometry of the present invention (Fig. 4 A and C) has produced how tangible spectrum than aluminium sheet (Fig. 4 B and D).From these results, find the application of the invention plate for mass spectrometry, can be simultaneously fast with in high sensitivity by mass spectrophotometry, the multiple proteins of analytical electrophoresis after separating.

Embodiment 4: the protein of analytical electrophoresis after separating electroblotting and in conjunction with the interaction between the albumen

As the application example of plate for mass spectrometry of the present invention, carried out following research: trace to the protein of plate for mass spectrometry and can with its generation interacting proteins between combine; Differentiate their protein complex subsequently by mass spectrophotometry.As for material, use SCUPA and antibody (anti-SCUPA antibody) thereof.

After SCUPA (4 μ g) is added into the 12%SDS-polyacrylamide gel, carried out electrophoresis 1 hour.After migration, the cutting gel is and in mode identical among the embodiment 3, on the plate for mass spectrometry with the preparation to the embodiment 1 of the SCUPA electroblotting on the gel.After the plate for mass spectrometry of blocking-up SCUPA trace, add antiserum (slightly refining, and contain anti-SCUPA antibody), and reaction is spent the night with ammonium sulfate.After finishing reaction, wash this plate with the PBS damping fluid, add matrix, carry out mass spectrophotometry.The results are shown among Fig. 5.

After the electrophoresis trace to the SCUPA of plate for mass spectrometry 48,616 places produce the peak, and observe and be fixed on SCUPA on the same plate and interactional anti-SCUPA antibody takes place at peak (Fig. 5 A) that 145,300 (73,115 are assigned to the divalent ion of same antibody) are located.In addition, in the gel that does not have SCUPA as a comparison, in this plate for mass spectrometry, do not observe the peak, although carried out identical process, for example electroblotting, blocking-up, the anti-SCUPA antibody of interpolation and with PBS washing (Fig. 5 B).

The above results has shown, after electrophoretic separation, the SCUPA of trace to the plate for mass spectrometry keep in conjunction with can with the activity of its generation interacting proteins (anti-SCUPA antibody), and measure in fact simultaneously trace protein and with the molecular weight of its generation interacting proteins, thereby can differentiate two kinds of molecular species; The above results has shown the protein-protein complex that can detect on this plate for mass spectrometry, and can differentiate the compound that forms on this plate for mass spectrometry.

Comparative example 1: trace is to pvdf membrane and mass spectrophotometry

Under reducing condition, on the 12%SDS-polyacrylamide gel, to two kinds of protein, promptly the potpourri series connection electrophoresis of each 4 μ g of SCUPA and HSA is twice.Add initial sample, carried out electrophoresis 40 minutes at the 30mA electric current, turn-off current is added into identical sample in the identical swimming lane once more then, further carries out electrophoresis 23 minutes under the 30mA electric current.After the migration, cut the gel of each migrating channels, further cut at the point of addition 39mm place on the gel upper surface; Shown in Fig. 2 A, the upper surface of two gels with them contacted separately and place, and the pvdf membrane of 78mm * 8mm cutting is placed on the gel, and in 10mM sodium borate buffer liquid (pH8.0), under the 90mA electric current, carried out electroblotting 2 hours.After finishing trace, wash pvdf membrane with PBS, use distilled water flushing, drying, and use the band (Sumitomo 3M Ltd. makes) of transparent double spread to be applied to aluminium sheet.Add matrix (DHB) to it, use ProteinChip System (aforesaid) to carry out mass spectrophotometry.

The result is, trace produces the peak to the SCUPA on the pvdf membrane at 50,096.9 places, and HSA produces the peak at 67,394.5 places; Yet, being difficult to identify this two peaks, this is because peak intensity is extremely low relatively, and signal to noise ratio (S/N ratio) (S/N ratio) low (Fig. 6).In addition, find (to be equally applicable to plate for mass spectrometry of the present invention) under the situation of plate for mass spectrometry, spend 2-3 minute and arrive vacuum state, it is feasible measuring immediately then; Yet, under the situation of pvdf membrane, reaches 45 minutes and arrive vacuum state, thereby cause employed mass spectrometer excessive load, and mass spectrometer is without undergoing frequent use.

Embodiment 5: use plate for mass spectrometry of the present invention to carry out the range protein group analysis

Use various cell or tissues to extract sample, (molecular weight 1,000-20,000) determines the performance of plate for mass spectrometry of the present invention in low relatively molecular weight ranges.In following test, sample is applied to 16%SDS-polyacrylamide gel (in the Tris-Tricine damping fluid), and electrophoresis 90 minutes, then in 10mM sodium borate buffer liquid (with hydrochloric acid with pH value regulated value 8.0) with analyte from gel electroblotting to stratographic analysis of the present invention with plate on 1-2 hour.After trace is finished, wash this plate, stain (spot) matrix, and carry out mass spectrophotometry.

(1) use 1N acetic acid homogenize pig cerebellum down at 4 ℃, and under 4 ℃, with 3, centrifugal 30 minutes of the rotating speed of 000rpm reclaims supernatant.Add acetonitrile, obtain final 10% concentration, supernatant is applied on the post of reverse-phase chromatography.Wash this post with 0.1% trifluoroacetic acid (TFA) that contains 10% acetonitrile, and with the 0.1%TFA wash-out that contains 60% acetonitrile.The freeze drying eluate, and used as the little brain extract of pig.After isolating extract by SDS-PAGE, with its electroblotting to plate for mass spectrometry of the present invention.Use contains the 0.5%TFA/50%ACN of saturated alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix, carries out mass spectrophotometry.The result is, 3, and 000-20, in 000 the molecular weight ranges, the peak is detectable.

Replace saturated CHCA to make comparisons with using DHB (150mg/mL ethanol) or saturated SPA (at 0.5%TFA/50%ACN) it as the situation of matrix.Under the situation of DHB, 3,000-20 in 000 the molecular weight ranges, does not observe tangible peak.Under the situation of saturated SPA, in identical molecular weight ranges, only observe several peaks.On the contrary, under the situation of saturated CHCA, 2,000-10,000 and 5,000-20 in 000 the molecular weight ranges, observes a large amount of peaks.From these results, show that CHCA more preferably is used to differentiate the protein in the relative low-molecular-weight scope.

(2) with above-mentioned (1) in identical mode, carry out the mass spectrophotometry of the little brain extract of pig, difference is: use 50% saturated CHCA as matrix.The result is, 1,000 or bigger molecular weight ranges in, the peak is detectable.Especially, detecting 3, during peak in the 000-20,000 molecular weight ranges, showing superior performance.

1, the degree aspect that the peak occurs in the mass spectrophotometry in the 000-10,000 molecular weight ranges replaces the situation of 50% saturated CHCA to make comparisons with using 10% or 20% saturated CHCA it.Under the situation of 10% saturated CHCA, observe several peaks.Under the situation of 20% saturated CHCA, the number at peak is slightly more than the situation of using 10% saturated CHCA.On the contrary, under the situation of 50% saturated CHCA, observe a lot of peaks.

(3) in the presence of 100ng/ml phorbol 12-myristinate 13-acetate (PMA), in RPMI 1640 media that contain 10%FCS, cultivate U937 cell (1 * 10 ⁵To 2 * 10 ⁶Individual cell/ml) 48 hours.Carry out identical experiment, but do not have a PMA, with in contrast.After cultivating end, with phosphate-buffered saline (PBS) washed cell, and preparation cell mass (cell pellet).In addition, add Protein Extraction reagent (Novagen Inc.) and protease inhibitors, after centrifuge cell group, under 4 ℃, left standstill cell mass 15 minutes, and reclaim supernatant, as cellular lysate.By the SDS-PAGE after separating, with its electroblotting to plate for mass spectrometry of the present invention.Subsequently, carry out mass spectrophotometry; 3, the intensity aspect at the detected peak in the 000-20,000 molecular weight ranges, relatively PMA stimulates the result of U937 cell and the result of contrast U937 cell.The result is to stimulate in the expression at the PMA of protein and observe following variation (table 1).

Table 1

The variation at peak (intensity)	The peak number order
		Disappear	38
Occur	11
		Reduce	1
Increase	2

(4) carry out the section mouse tissue.Use Beads Shocker (Yasui Kikai Corporation, Osaka), 2, the 500rpm rotating speed is handled 10-30 second down, the mouse tissue (brain, lung, liver, muscle) that breaks adds TricinePAGE sample damping fluid (Wako Pure Chemical Industries), 12, under the 000rpm centrifugal 5 minutes, reclaim supernatant.Using the SDS-PAGE after separating, the supernatant electroblotting to plate for mass spectrometry of the present invention, is being carried out mass spectrophotometry.The result is to detect tissue-specific peptide (molecular weight 3,000-20,000) in each tissue.

Embodiment 6: the influence of damping fluid during the research electroblotting

Damping fluid during as electroblotting, use Tris damping fluid (25mM Tirs/192mM glycocoll/20% methyl alcohol, pH8.3), phosphate buffer (5%ACN/125mM NaCl/PBS, pH7.2), acetate buffer (40mM Tris-acetate/1mM EDTA, pH8.0) and borate buffer solution (10mM sodium borate-hydrochloric acid, pH8.0).Protein as for mass spectrophotometry is used uses HSA and SCUPA.Except these conditions, the method according to embodiment 5 experimentizes.The result is that in the damping fluid that detects, borate buffer solution produces maximum mass spectra peak intensity.For example, for SCUPA, the maximal value of the peak intensity of borate buffer solution is 21 times of phosphate buffer approximately, for HSA, and about 4 times.

Industrial applicibility

Because use PVDF as the part adsorbent, plate for mass spectrometry of the present invention can adsorb range protein equably, excellent on trace efficient, and the normally three-dimensional structure and the function of retaining protein.The feature of plate for mass spectrometry of the present invention also is: do not have non-specific adsorption, carry out the ability of mass spectrophotometry etc. behind the trace.

Utilize the feature of plate for mass spectrometry of the present invention, can analyze and differentiate protein in a large number, fast and in high sensitivity.Protein can be a kind of protein, the perhaps aggregate of multiple proteins (protein group), even can analyze and differentiate the compound (that is to say, a large amount of, fast and in high sensitivity) that this protein is had the compound of affinity (with its interaction) equally.

In addition, can significantly reduce the running time, step is simplified, plant bulk reduces, cost reduction, robotization and screening macromethod thing, and make the analysis of large-scale protein matter easy.And, can the important low abundance proteins of Proteomic analysis physiology, thus the seed compound (seed compound) that helps to find the biomarker of diagnostic uses and find the novel drugs research and development.

Therefore, the invention enables and can introduce a kind of new technology in traditional Proteomic analysis, it is the combination of electrophoresis and mass spectrophotometry.

The application is based on to the US 10/264,505 of U. S. application with to the Japanese patent application No.2002-344710 of Japanese publication, whole in this manual instruction of introducing this two applications.

In this instructions, introduce all references, comprise publication and patent referred in this, by at this to show the degree that is introduced into separately as them, as a reference, in them all in this qualification.

Although emphasis has been described the present invention with preferred embodiment, it is apparent to those skilled in the art that these preferred embodiment can change.The present invention can realize by the method except that the method for describing in detail herein.Therefore, the present invention includes the various changes of including in the spirit and scope of claim.

Claims (16)

1. plate for mass spectrometry, it comprise supporter and with the coating of its adhesion, wherein said coating contains polyvinylidene fluoride.

2. the plate of claim 1, wherein supporter is made by aluminium or stainless steel.

3. the method for preparing plate for mass spectrometry, it comprises with polyvinylidene fluoride and is coated with supporter.

4. the method for claim 3, wherein Tu Bu mode be smear, injection, vapour deposition, dipping, printing or sputter.

5. claim 3 or 4 method, it comprises that the solution that will contain polyvinylidene fluoride is applied to supporter.

6. the method for claim 5 is wherein used solvent in described solution, and described method comprise use after, remove described solvent.

7. plate for mass spectrometry, it is by each method preparation among the claim 3-6.

8. the method for discriminatory analysis thing, it comprises the steps (a)-(d):

(a) provide the plate for mass spectrometry of claim 1 or 7,

(b) sample that contains analyte is carried out gel electrophoresis,

(c) the gel trace after will moving is to described plate, so that described analyte is transferred on the described plate, and

(d) described plate is carried out mass spectrophotometry, to analyze the analyte of transfer printing.

9. the method for claim 8, wherein trace is an electroblotting.

10. claim 8 or 9 method, wherein mass spectrophotometry is MALDI-MS.

11. the method for claim 8, the sample that wherein contains analyte contains multiple analytes.

12. the method for claim 8, wherein analyte is selected from protein, nucleic acid, oligonucleotides, sugar, oligosaccharides, cell-membrane receptor activator or antagonist, toxin, virus epitopes, hormone, peptide, enzyme, zymolyte or enzyme inhibitor, co-factor, medicine, agglutinin and antibody.

13. the method for claim 8, further be included in step (c) and (d) between step (c2), the sample that wherein makes the analyte that contains transfer printing have the material of affinity contacts with described plate, to form the compound of described analyte and described material onboard, in step (d), analyze described analyte and described material thus simultaneously.

14. the method for claim 8 further comprises: between step (c) and step (d), mass spectrophotometry is added into described plate with matrix.

15. the method for claim 13 further comprises: between step (c2) and step (d), mass spectrophotometry is added into described plate with matrix.

16. the method for claim 14 or 15, its mesostroma is 2, the 5-dihydroxy-benzoic acid.

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