High Throughput Peptide Mass Fingerprinting and Protein Macroarray Analysis Using Chemical Printing Strategies
2002; Elsevier BV; Volume: 1; Issue: 7 Linguagem: Inglês
10.1074/mcp.m200020-mcp200
ISSN1535-9484
AutoresAndrew J. Sloane, Janice L. Duff, Nicole L. Wilson, Parag S. Gandhi, Cameron J. Hill, Femia G. Hopwood, Paul E. Smith, Mélissa Thomas, Robert A. Cole, Nicolle H. Packer, Edmond J. Breen, Patrick W. Cooley, David B. Wallace, Keith L. Williams, Andrew A. Gooley,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoWe describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery. We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery. The ability to accurately define protein expression in relationship to physiological changes associated with healthy or diseased states and the potential to discover novel drug targets are emerging themes of proteomic programs (1.Abbott A. A post-genomic challenge: learning to read patterns of protein synthesis.Nature. 1999; 402: 715-720Google Scholar, 2.Banks R.E. Dunn M.J. Hochstrasser D.F. Sanchez J.C. Blackstock W. Pappin D.J. Selby P.J. Proteomics: new perspectives, new biomedical opportunities.Lancet. 2000; 356: 1749-1756Google Scholar). Understanding these dynamics is rendered complex given there is often no correlation between mRNA expression levels and protein expression (3.Anderson L. Seilhamer J. A comparison of selected mRNA and protein abundances in human liver.Electrophoresis. 1997; 18: 533-537Google Scholar), and the paradigm of one gene-one protein is known not to hold (4.Wilkins M.R. Sanchez J.C. Williams K.L. Hochstrasser D.F. 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Sample prefractionation, advances in solubilization strategies, and improvements in two-dimensional gel electrophoresis (2-DE) 1The abbreviations used are: 2-DE, two-dimensional gel electrophoresis; BSA, bovine serum albumin; CFR, curved field reflectron; ESI, electrospray ionization; FITC, fluorescein isothiocyanate; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; OGP, n-octyl β-d-glucopyranoside; PBS-TAC, phosphate-buffered saline containing 0.1% (v/v) Tween 20, 0.05% (w/v) NaN3, and 0.5% (w/v) casein, pH 7.4; pmf, peptide mass fingerprinting; PSL, Proteome Systems Limited; PVDF, polyvinylidene fluoride; TB, tuberculosis. are further refining this art (6.Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9390-9395Google Scholar, 7.Herbert B. 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Generation of a baculovirus recombinant prostate-specific membrane antigen and its use in the development of a novel protein biochip quantitative immunoassay.Protein Expr. Purif. 2000; 19: 12-21Google Scholar) for screening complex protein mixtures for binding affinities, protein associations, and disease markers. With a move toward automation, deposition techniques used to produce these arrays now include pin-based or microdispensing liquid-handling robots (14.Lueking A. Horn M. Eickhoff H. Bussow K. Lehrach H. Walter G. Protein microarrays for gene expression and antibody screening.Anal. Biochem. 1999; 270: 103-111Google Scholar), photolithography (26.Mooney J.F. Hunt A.J. McIntosh J.R. Liberko C.A. Walba D.M. Rogers C.T. Patterning of functional antibodies and other proteins by photolithography of silane monolayers.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12287-12291Google Scholar, 27.Jones V.W. Kenseth J.R. Porter M.D. Mosher C.L. Henderson E. Microminiaturized immunoassays using atomic force microscopy and compositionally patterned antigen arrays.Anal. Chem. 1998; 70: 1233-1241Google Scholar), and ink-jet printing technology (28.Cooley P. Hinson D. Trost H.J. Antohe B. Wallace D. Ink-jet-deposited microspot arrays of DNA and other bioactive molecules.Methods Mol. Biol. 2001; 170: 117-129Google Scholar, 29.Hughes T.R. Mao M. Jones A.R. Burchard J. Marton M.J. Shannon K.W. Lefkowitz S.M. Ziman M. Schelter J.M. Meyer M.R. Kobayashi S. Davis C. Dai H. He Y.D. Stephaniants S.B. Cavet G. Walker W.L. West A. Coffey E. Shoemaker D.D. Stoughton R. Blanchard A.P. Friend S.H. Linsley P.S. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.Nat. Biotechnol. 2001; 19: 342-347Google Scholar, 30.Roda A. Guardigli M. Russo C. Pasini P. Baraldini M. Protein microdeposition using a conventional ink-jet printer.Biotechniques. 2000; 28: 492-496Google Scholar). Despite advantages of protein array technology such as speed, sensitivity, and multi-screening capabilities (15.Cahill D.J. Protein and antibody arrays and their medical applications.J. Immunol. Methods. 2001; 250: 81-91Google Scholar), chip-based proteomics has the major caveat that the protein arrays are either constructed from known proteins, such as antibodies, or an array of proteins derived from a recombinant expression system (15.Cahill D.J. Protein and antibody arrays and their medical applications.J. Immunol. Methods. 2001; 250: 81-91Google Scholar). Hence, the current chip-based approaches ignore the many protein isoforms produced by cells such as different co- and post-translationally modified forms of the same translated gene product, members of gene families, and variable spliced variants of mRNA and protein. Recent advances in sample preparation have enabled many of the known protein isoforms to be displayed in 2-DE arrays (8.Herbert B. Righetti P.G. A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes.Electrophoresis. 2000; 21: 3639-3648Google Scholar). A Western blot of 2-DE separated proteins onto a membrane such as polyvinylidene fluoride (PVDF) or nitrocellulose represents a protein chip, albeit a macroarray. The protein macroarray differs from a protein chip microarray, because the coordinates of each protein are determined by the physical attributes of the isoelectric point and apparent molecular weight of the protein. Once the coordinates of each protein within the protein macroarray are identified by an image-capture device, each protein spot then has a defined X and Y position and can be manipulated by robotic platforms. Here we present technology that combines the advantages of both protein chips and 2-DE, which we have described as a chemical printer (Fig. 1). The chemical printer uses a microjet device utilizing piezoelectric drop-on-demand-type ink-jet technology for rapid liquid microdispensing (31.Adams R.L. Roy J. A one-dimensional numerical model of a drop-on-demand ink jet.J. Appl. Mech. 1984; 53: 193-197Google Scholar, 32.Bogy D.B. Talke F.E. Experimental and theoretical study of wave propagation phenomena in drop-on-demand ink jet devices.IBM J. Res. Dev. 1984; 29: 314-321Google Scholar, 33.Dijksman J.F. Hydrodynamics of small tubular pumps.J. Fluid Mech. 1984; 139: 173-191Google Scholar). The ability of ink-jet printing to dispense minimal amounts of rare fluids and permit parallel processing of large numbers of tests means assay sizes can be decreased whereas assay density is increased (34.Ekins R. Immunoassay: recent developments and future directions.Nucl. Med. Biol. 1994; 21: 495-521Google Scholar, 35.Wallace D.B. Cox W.R. Hayes D.J. Piqué A. Chrisey D.B. Direct-write Technologies for Rapid Prototyping of Sensors, Electronics, and Passivation Coatings. 1st Ed. Academic Press, San Diego2001: 213-220Google Scholar). These qualities have lead to increased applications for dispensing of bioactive materials. Immunodiagnostics and antibody/antigen dispensing (36.Hayes, D. J., Wallace, D. B., VerLee, D., and Houseman, K. (October 31, 1989) U. S. Patent 4,877,745Google Scholar), synthesis and deposition of oligonucleotides in microarray formats (29.Hughes T.R. Mao M. Jones A.R. Burchard J. Marton M.J. Shannon K.W. Lefkowitz S.M. Ziman M. Schelter J.M. Meyer M.R. Kobayashi S. Davis C. Dai H. He Y.D. Stephaniants S.B. Cavet G. Walker W.L. West A. Coffey E. Shoemaker D.D. Stoughton R. Blanchard A.P. Friend S.H. Linsley P.S. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.Nat. Biotechnol. 2001; 19: 342-347Google Scholar, 38.Goldmann T. Gonzalez J.S. DNA printing: utilization of a standard inkjet printer for the transfer of nucleic acids to solid supports.J. Biochem. Biophys. Methods. 2000; 42: 105-110Google Scholar, 39.Okamoto T. Suzuki T. Yamamoto N. Microarray fabrication with covalent attachment of DNA using bubble jet technology.Nat. Biotechnol. 2000; 18: 438-441Google Scholar), protein and peptide analysis (40.Mueller U. Nyarsik L. Horn M. Rauth H. Przewieslik T. Saenger W. Lehrach H. Eickhoff H. Development of a technology for automation and miniaturization of protein crystallization.J. Biotechnol. 2001; 85: 7-14Google Scholar), and drug discovery (41.Lemmo A.V. Rose D.J. Tisone T.C. Inkjet dispensing technology: applications in drug discovery.Curr. Opin. Biotechnol. 1998; 9: 615-617Google Scholar) are a number of applications utilizing ink-jet printing technologies. We demonstrate in situ proteinase digests of membrane-immobilized protein macroarrays and subsequent MALDI-TOF MS analysis directly from the membrane surface. This approach bypasses the multiple liquid-handling steps associated with in-gel digestion procedures. Importantly, the ability to analyze immobilized proteins allows for both archiving of samples pre-/post-analysis and for multiple chemical reactions to be performed at different locations on an individual protein spot. We demonstrate identification of N-linked glycosylation sites by MALDI-TOF MS using sequential PNGase F and trypsin digestion, as well as preparation and extraction of oligosaccharides from PVDF membrane for structural analysis by LC-ESI MS. An exciting new high throughput assay also uses the chemical printer to microdispense antibodies onto membrane-immobilized proteins to rapidly define immunoreactivity and quantitate signals in a solid phase enzyme-linked immunosorbent assay-like format. The chemical printer thus represents a powerful tool for identification of novel protein targets for biomedical and diagnostic purposes. Standard laboratory chemicals were obtained from Sigma unless specified otherwise. Human serum and plasma samples and 38 kDa tuberculosis (TB) protein were gifts from AP Clinical (Sydney, Australia). Where stipulated in the text, human serum albumin was depleted from plasma using methods described previously (42.Cohn E.J. Strong L.E. Hughes Jr., W.L. Mulford D.J. Ashworth J.N. Melin M. Taylor H.L. Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein lipoprotein components of biological tissues and fluids.J. Am. Chem. Soc. 1946; 68: 459-475Google Scholar, 43.Oncley J.L. Melin M. Richert D.A. Cameron J.W. Gross Jr., P.M. The separation of the antibodies, isoagglutinins, prothrombin, plasminogen and β1-lipoprotein into sub-fractions of human plasma.J. Am. Chem. Soc. 1949; 71: 541-550Google Scholar). Human platelets were purchased from the Red Cross Blood Bank (Sydney, Australia). Contaminating red blood cells were removed from the platelets by centrifugation at 200 × g for 10 min at 4 °C. The platelet-rich plasma was then centrifuged at 1500 × g for 20 min at 4 °C. The platelet component of the pellet was removed gently and then resuspended in 50 mm Tris-HCl, 5 mm EDTA, pH 7.4. The platelets were washed similarly two more times. Whole platelets were finally solubilized using a ProteoPrep™ sample extraction kit (Sigma) using the supplied cellular and organelle membrane solubilizing reagent, to a final protein concentration of 4 mg/ml. Purified immunoglobulin was obtained from CSL (Parkville, Australia). Solutions were dispensed using an α-version chemical printer being developed by Proteome Systems Ltd. (PSL) (Sydney, Australia) in collaboration with Shimadzu Biotech (Kyoto, Japan). Solutions were pre-filtered through either 0.22- or 0.45-μm membrane filters (Millipore). Glass capillary piezoelectric microjet devices (Microfab Technologies, Inc., Plano, TX) were used to dispense all solutions. X and Y coordinates of target protein spots were determined using ImagepIQ™, an in-house image-capture product (PSL). 36 μl of whole human plasma was made up to a final volume of 490 μl in 7 m urea, 2 m thiourea, 2% (w/v) CHAPS, and 5 mm Tris, pH 10.2. The sample was then ultrasonicated for 30 s, reduced with 3 mm tributylphosphine for 2 h, and then alkylated with 15 mm iodoacetamide for 1 h. Before rehydration of immobilized pH gradient strips, sample was ultrasonicated for 2 min and then centrifuged at 21000 × g for 5 min. The supernatant was collected, and 10 μl of Orange G was finally added as an indicator dye. Sample prefractionation into narrow range pI fractions was performed with an ElectrophoretIQ™ multicompartment electrolyzer (8.Herbert B. Righetti P.G. A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes.Electrophoresis. 2000; 21: 3639-3648Google Scholar) (PSL). Dry 11-cm immobilized pH gradient strips (Amersham Biosciences) were rehydrated for 6 h with 200 μl of protein sample. Rehydrated strips were focused on a Protean IEF cell (Bio-Rad) or PSL prototype IsoElectrIQ™ electrophoresis equipment for 120 kV-h at a maximum of 10 kV. Focused immobilized pH gradient strips were equilibrated for 20 min in 6 m urea, 2% (w/v) SDS, 50 mm Tris-HCl, pH 7.0. Equilibrated strips were inserted into loading wells of 6–15% (w/v) Tris acetate SDS-PAGE pre-cast prototype 10 × 15-cm GelChips™ (PSL). Electrophoresis was performed at 50 mA for 1.5 h. Proteins were electroblotted onto 0.45-μm nitrocellulose (Bio-Rad) or Immobilon PSQ PVDF membranes (Millipore) using a prototype ElectrophoretIQ™ electroblotting apparatus (PSL) at 400 mA for 1.3 h and methods described by Kyhse-Andersen (44.Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose.J. Biochem. Biophys. Methods. 1984; 10: 203-209Google Scholar). Proteins were finally stained using Direct Blue 71. Titrated amounts of BSA were prepared in SDS-PAGE sample buffer containing 2% (w/v) SDS, 20% (v/v) glycerol, 0.025% (w/v) bromphenol blue, 50 mm dithiothreitol, 10 mm acrylamide, 0.375 m Tris, pH 8.8. Samples were allowed to reduce and alkylate for 1 h at room temperature prior to electrophoresis using a 6–15% (w/v) polyacrylamide ProteoGel™ (Sigma) and the conditions described above. Protein was finally electrotransferred onto an Immobilon PSQ PVDF membrane as described above. Immobilon PSQ PVDF membranes were first adhered to an Axima-CFR MALDI-TOF target plate (Kratos, Manchester, United Kingdom) using 3M™ electrically conductive tape 9703 (St. Paul, MN). Porcine trypsin (Promega, Madison, WI) at 200 μg/ml in 25 mm NH4HCO3, pH 8.5, or Staphylococcus aureus V8 endoproteinase Glu-C (Roche Molecular Biochemicals) at 200 μg/ml in 5 mm NaHPO4, pH 7.8, were dispensed as 50 or 25 iterations, respectively, onto protein spots at 1 nl per iteration. Prior to dispensing trypsin or Glu-C, either 3 × 1.5-nl iterations of 1% (v/v) n-octyl β-d-glucopyranoside (OGP) or 5 nl of 1% (w/v) polyvinylpyrrolidone (PVP40) in 50% (v/v) methanol, respectively, were printed to pre-wet the PVDF membrane. Excess PVP40 was removed by washing with water using a transfer pipette. Digestion was performed for 3 h at 37 °C in a humidified environment. After digestion, 50 × 2-nl iterations of 10 mg/ml matrix solution, α-cyano-4-hydroxycinnamic acid in a methanol/2-propanol/2-butanol/0.5% (v/v) formic acid solution was then dispensed on top of the digestion zone of each spot. Digests were analyzed using an Axima-CFR MALDI-TOF mass spectrometer (Kratos). All spectra underwent an internal two-point calibration using autodigested trypsin peak masses, m/z 842.51 and 2211.10 Da. Software designed by PSL was used to resolve isotopic peaks from MS spectra (45.Breen E.J. Hopwood F.G. Williams K.L. Wilkins M.R. Automatic poisson peak harvesting for high throughput protein identification.Electrophoresis. 2000; 21: 2243-2251Google Scholar). In-house databases and tools (PSL) and PeptIdent from the ExPASy molecular biology server (www.expasy.ch/tools/peptident.html) were used for pmf analysis using a mass tolerance of 100 ppm. Protein gel pieces were excised using a prototype Xcise™ system (PSL and Shimadzu Biotech) and then washed with 25 mm NH4HCO3, pH 8.5. Gel pieces were then dehydrated under vacuum for 15 min and digested with 10 μl of 20 μg/ml porcine trypsin in 25 mm NH4HCO3, pH 8.5, for 3 h at 37°C. Peptides were extracted from gel pieces with 10 μl of 50% (v/v) acetonitrile, 0.5% (v/v) formic acid and sonication for 10 min. Prior to MALDI-TOF MS analysis, peptides were concentrated and purified using a C18 ZipTip® (Millipore) and eluted onto a target plate in 2 μl of matrix solution and allowed to dry. N-linked oligosaccharides were cleaved on the PVDF membrane, which was adhered to a MALDI-TOF plate by printing 50 × 10-nl iterations of 5 units/μl PNGase F (Roche Molecular Biochemicals) per protein spot and incubation for 3 h at 37°C in a humidified environment. Released oligosaccharides were extracted into 1 μl of water by pipette for LC-ESI MS analysis. The membrane was then washed with water using a transfer pipette. PNGase F-treated protein was subsequently digested on the membrane with trypsin using chemical printing followed by MALDI-TOF MS analysis. Sample was loaded onto a ThermoHypersil 5-μm Hypercarb column (Keystone Scientific Operations, Bellefonte, PA) using a Surveyor autosampler connected to an LCQ Deca mass spectrometer (ThermoFinnigan, San Jose, CA). Oligosaccharides were separated and eluted using a 30-min 0–25% (v/v) acetonitrile gradient in 10 mm NH4HCO3. Tandem MS analysis was performed in negative ion mode over a m/z range of 320 to 2000 Da. Oligosaccharide structures were predicted using the GlycoSuite™ database (PSL). Human serum was diluted 1:3 with phosphate-buffered saline containing 0.1% (v/v) Tween 20, 0.05% (w/v) NaN3, and 0.5% (w/v) casein, pH 7.4 (PBS-TAC), and then filtered through a 0.22-μm membrane (Millipore). Five applications of 10 nl of serum were printed onto proteins immobilized on a nitrocellulose membrane after blocking nonspecific binding sites with PBS-TAC for 15 min at room temperature. The membrane assay area was clamped onto a layer of absorbent tissue paper to prevent dispersal of fluid over the membrane surface during microdispensing. Excess antibody was removed by washing twice with 2 × 50-μl drops of PBS-TAC using a transfer pipette. Bound antibody was detected by pipetting 20 μl of goat anti-human IgG conjugated to fluorescein isothiocyanate (FITC) (Zymed Laboratories Inc., San Francisco, CA) diluted 1/10 with PBS-TAC. Fluorescence was detected using a Bio-Rad FluorS™ system. Conventional pmf analysis of purified proteins involves isolation of individual protein spots from a gel (or membrane) followed by in-gel tryptic digestion, peptide extraction, peptide clean up, and finally loading the extracts onto a MALDI-TOF target (46.Henzel W.J. Billeci T.M. Stults J.T. Wong S.C. Grimley C. Watanabe C. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5011-5015Google Scholar). As an alternative rapid high throughput approach, we demonstrate dispensing of trypsin onto a macroarray of human plasma proteins on a PVDF membrane that had been adhered to a MALDI-TOF target plate (Fig. 2A). The most efficient digestion conditions were achieved by jetting 200 μg/ml trypsin in 50 × 1-nl iterations onto each protein spot. The small amount of drying time between each iteration increased the digestion efficiency by preventing excessive diffusion of trypsin solution across the membrane surface. Digestion sites on the hydrophobic PVDF membrane surface were pre-wet by printing either 4.5 nl of the non-ionic detergent nI-octyl β-d-glucopyranoside or 5 nl of PVP40. This also prevented nonspecific binding of proteinase to the membrane. After digestion, matrix solution was dispensed directly onto the tryptic peptides prior to MALDI-TOF MS analysis directly from the membrane surface. Fig. 2A illustrates the relative size of the digestion zones (∼200–300-μm diameter). Of 14 protein spots representing a range of proteins present in various amounts (Fig. 2A) all were identified successfully by pmf analysis after on-membrane digestion (Table I). Fig. 2B shows a representative spectrum of apolipoprotein E generated after on-membrane digestion.Table IComparison of pmf analysis of proteins identified from on-membrane versus in-gel tryptic digestionsSpot numberProtein nameAccession numberIn-gel digestOn-membrane digestPeptide hits% CoveragePeptide hits% Coverage1α-1-AntitrypsinP010092762.41131.72Antithrombin-IIIP010081643.81631.03Apolipoprotein A-IVP067272664.12253.74Haptoglobin-1 β-chainP007372260.01239.2Haptoglobin-2 β-chainP007382260.01239.25Fibrinogen γ-chainP044692464.61834.46Serum albuminP027682544.83245.07Serotransferrin chain 1P027873355.43248.28Fibrinogen β chainP026751538.82253.59Apolipoprotein EP026492781.32874.910Serum amyloid P-componentP02743835.31034.811Apolipoprotein A-IP026472079.4929.612Apolipoprotein A-IP026472684.41139.513Ig κ chain C regionP01834567.0876.414TransthyretinP027661188.2865.415Pyruvate kinaseP146182044.11520.516Fructose bisphosphate aldolaseP040751453.2620.117Fructose bisphosphate aldolaseP040751246.3825.618Glyceraldehyde 3-phosphate dehydrogenaseP044061348.21746.119Peptidyl prolyl cis-trans isomerase chain 1 (cyclophilin A)P23284628.4418.620Nucleotide diphosphate kinase BP22392962.5536.2 Open table in a new tab Detection of low abundance proteins is often a limitation in proteomic studies. Furthermore, alkaline proteins are even more difficult to identify given the difficulty in focusing such highly positive charged proteins. For this reason we have analyzed several low abundance alkaline platelet proteins to demonstrate that identification of such proteins is achievable using on-membrane digestion methods with the chemical printer (see Fig. 3 and Table I). Positive identification of nucleoside diphosphate kinase B (Table I) was confirmed following post-source decay analysis of the 1175.76 ion (data not shown). The sequence coverages from peptides generated by on-membrane digestion were usually less than those from in-gel digests (Table I). This was not unexpected given the in-gel digests were each purified using a C18 ZipTip®, and the excised area of the in-gel digest was 1.2 mm in diameter, more than five times the area digested on-membrane. In some cases increased numbers of peptide hits were observed off the membrane, but lower sequence coverages were obtained relative to in-gel digests. This was a result of the higher abundance of smaller peptides extracted from the membrane surface (Table I). Nevertheless, with respect to sensitivity of peptide detection, analysis of on-membrane tryptic digests of BSA demonstrated that a targeted protein amount of 10 fmol of BSA was still sufficient for generating pmf data that enabled reliable protein identification (Table II).Table IIMALDI-TOF MS sensitivity for detection of on-membrane tryptic peptides of BSATotal amount of BSA per laneApproximate amount of BSA digestedPeptide hits%Coveragefmol250025014 ± 426.8 ± 5.9100010020 ± 534.4 ± 8.05005013 ± 223.1 ± 2.72502510 ± 319.8 ± 6.5100109 ± 317.5 ± 6.1 Open table in a new tab
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