Selective Detection of Membrane Proteins Without Antibodies
2002; Elsevier BV; Volume: 1; Issue: 2 Linguagem: Inglês
10.1074/mcp.m100027-mcp200
ISSN1535-9484
AutoresDavid Arnott, Adrianne Kishiyama, Elizabeth Luis, Sarah G. Ludlum, James C. Marsters, John T. Stults,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoA method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude. A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude. Tools for the measurement and analysis of gene and protein expression patterns are at the core of several recently defined disciplines, functional genomics, transcriptomics, proteomics, and subfields such as pharmacogenomics and pharmacoproteomics. Among these tools, differential display of mRNA is performed routinely using cDNA microarrays (1Schena M. Shalon D. Heller R. Chai A. Brown P.O. Davis R.W. Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10614-10619Google Scholar, 2Li S. Rose D.T. Kadin M.E. Brown P.O. Wasik M.A. Comparative genome-scale analysis of gene expression profiles in T cell lymphoma cells during malignant progression using a complementary DNA microarray.Am. J. Pathol. 2001; 158: 1231-1237Google Scholar). Fluorescence detection, together with the amplification of DNA using the polymerase chain reaction, allows such experiments to be performed with exquisite sensitivity, and the parallel detection of thousands of gene products enables high throughput measurements. For protein measurement, 2D 1The abbreviations used are: 2D, two-dimensional; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; CID, collision-induced dissociation; ICAT, isotope-coded affinity tag; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PSCA, prostate stem cell antigen; HPLC, high pressure liquid chromatography. PAGE is capable of resolving 2500 or more distinct protein spots (3Celis J. Gromov P. 2D protein electrophoresis, can it be perfected?.Curr. Opin. Biotechnol. 1999; 10: 16-21Google Scholar), making it the highest resolution protein separation experiment yet devised. This venerable technique has undergone a rebirth because of advances in reproducibility and automation and the ability to identify most detectable proteins using mass spectrometry and sequence data base searching (4Lopez M.F. Proteome analysis I. Gene products are where the biological action is.J. Chromatogr. B. 1999; 722: 191-202Google Scholar, 5Humphrey-Smith I. Cordwell S.J. Blackstock W.P. Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.Electrophoresis. 1997; 18: 1217-1242Google Scholar). More recently, a particularly powerful technique to emerge is the combination of liquid chromatography and mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS). Because spectra from only a few peptides (or even a single peptide) can be sufficient to identify a protein, multiple components of a protein mixture can be identified (6Arnott D. Henzel W.J. Stults J.T. Rapid identification of comigrating proteins by ion trap-mass spectrometry.Electrophoresis. 1998; 19: 968-980Google Scholar). Several groups have used this technology to identify hundreds of proteins from the tryptic digests of crude cellular extracts (7Davis M.T. Beierle J. Bures E.T. McGinley M.D. Mort J. Robinson J.H. Spahr C.S. Yu W. Luethy R. Patterson S.D. Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses.J. Chromatogr. B. 2001; 752: 281-291Google Scholar, 8Link A.J. Eng J. Scheiltz D.M. Carmack E. Mize G.J. Morris D.R. Garvick B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar, 9Spahr C.S. Susin S.A. Bures E.J. Robinson J.H. Davis M.T. McGinley M.D. Kroemer G. Patterson S.D. Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides.Electrophoresis. 2000; 21: 1635-1650Google Scholar, 10Davis M.T. Lee T.D. Variable flow liquid chromatography-tandem mass spectrometry and the comprehensive analysis of complex protein digest mixtures.J. Am. Soc. Mass Spectrom. 1997; 8: 1059-1069Google Scholar, 11Opiteck G.J. Ramirez S.M. Jorgenson J.W. Moseley III, M.A. Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping.Anal. Biochem. 1998; 258: 349-361Google Scholar, 12Washburn M.P. Wolters D. Yates III, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Google Scholar). The isotope-coded affinity tag methodology (ICAT) first described by Gygi and colleagues (13Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Google Scholar, 14Griffin T.J. Gygi S.P. Rist B. Aebersold R. Loboda A. Jilkine A. Ens W. Standing K.G. Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer.Anal. Chem. 2001; 73: 978-986Google Scholar) has extended such experiments to allow relative quantitation of proteins between two samples. This technique involves differential labeling of proteins in two samples with affinity (e.g. biotinylation) reagents differing slightly in mass. After mixing and digestion of the samples the labeled peptides are isolated by affinity chromatography and analyzed by mass spectrometry. Each peptide is detected as two peaks in an LC-MS experiment. Tandem MS is used to identify the protein from which each peptide is derived, and the relative abundances of corresponding peaks reflect the amounts of protein in each sample from which they were derived. As powerful and complementary as current genomic and proteomic tools are, they nevertheless suffer several shortcomings. Although tools such as cDNA microarrays are extraordinarily powerful for the simultaneous detection of thousands of gene products, mRNA levels do not necessarily correlate with protein expression levels (15Anderson L. Seilhamer J. A comparison of selected mRNA and protein abundances in human liver.Electrophoresis. 1997; 18: 533-537Google Scholar, 16Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Google Scholar). 2D PAGE is of limited use in verifying DNA microarray results, because it is difficult to predict in advance which of the potentially thousands of spots corresponds to a given protein because of the spectrum of possible post-translational modifications. Alternatively, fluorescence-activated cell sorting, immunohistochemistry, Western blots, enzyme-linked immunosorbent assays, and other antibody-based approaches can be used to explore the expression patterns and biological function of proteins. These powerful, but often time-consuming, techniques are currently the methods of choice to expand on the results of mRNA-based experiments. Reliance on antibodies, however, makes this difficult to do quickly, because antibodies must first be generated for each target protein. Furthermore, an antibody that binds a native protein (as in immunoprecipitation) may not be useful for detecting the denatured protein on a Western blot. Thus, a technique is needed that is similar to a Western blot but does not require an antibody to each protein of interest. Such a technique should be rapid, sensitive, quantitative, and capable of identifying a specific protein out of extremely complex mixtures without bias or need for extensive purification of intact proteins. We have developed an analytical procedure with the potential to meet these requirements based on the ICAT methodology of Gygi et al. (13Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Google Scholar). This experiment, dubbed the mass western in analogy to the Western blot, was applied to the detection of proteins from plasma membrane preparations of human cells without electrophoresis or other initial purification steps. Proteins assayed included the prostate stem cell antigen (PSCA) and the receptor tyrosine kinase ErbB2, which are over-expressed in significant numbers of prostate tumor and breast tumors, respectively. Breast tumor cell lines SK-BR-3 and MCF-7, or prostate tumor cell line PC3 transfected with the full-length sequence of PSCA, were used in these experiments. Eighty million of the transfected PC3 cells (designated clone 11) were used in the PSCA experiment. Fifty million MCF-7 cells and approximately half as many SK-BR-3 cells, normalized by total protein content, were used in the ErbB2 study. Cells cultured in flasks (175 cm2; Falcon) were harvested and disrupted with a Dounce homogenizer (17Graham J.M. Graham J. Higgins J. Biomembrane Protocols I. Isolation and Analysis. Vol. 19. Humana Press, Totowa, NJ1993: 97-108Google Scholar). In the ErbB2 experiment the cell lysate was centrifuged at 1000 × g for 5 min to remove intact cells and nuclei. The resulting post-nuclear supernatant was centrifuged at 16,000 × g for 15 min to produce a crude membrane pellet. In the PC-3 experiment the post-nuclear supernatant was layered over a 35% (w/v) sucrose solution and centrifuged in a SW55Ti rotor at 38,000 × g for 45 min. The membrane layer was collected, pelleted, and washed successively for 30 min with each of the following: 1) ice-cold 25 m m Na2CO3, pH 11, to remove cytoskeletal proteins (18Hubbard A.L. Ma A. Isolation of rat hepatocyte plasma membranes II, identification of membrane-associated cytoskeletal proteins.J. Cell Biol. 1983; 96: 230-239Google Scholar), 2) 0.5% Tween 20 (Calbiochem) at 4 °C, and 3) cold phosphate-buffered saline, with each step followed by centrifugation at 16,000 × g for 15 min. Protein concentrations were determined by bicinchonic acid assay. α-N-iodoacetyl-ε-N-biotinyl- l-lysine trideuteromethyl ester was prepared in a two-step synthesis. ε-N-biotinyl-l-Lysine (biocytin, 100 mg; Sigma) was suspended in methanol-d4 (2 ml; Cambridge Isotope Laboratory) and 4 n HCl/dioxane (2 ml; Pierce) in a sealed flask overnight, evaporated to dryness, and evaporated twice more from toluene. The resulting trideuteromethyl ester was used without further purification. The residue was dissolved in N,N-dimethylformamide (1 ml) and N,N-diisopropylethylamine (0.2 ml; Aldrich) and treated with iodoacetic anhydride (114 mg; Aldrich). After 30 min, the reaction mixture was loaded directly onto a C18 HPLC prep column (21 × 250 mm; Dynamax) in 100 ml of 5% acetonitrile/0.1% trifluoroacetic acid/water and eluted using a acetonitrile gradient (5–29%, 0.1% trifluoroacetic acid). Fractions were analyzed via electrospray mass spectrometry (API-1; Sciex) and combined and lyophilized to yield α-N-iodoacetyl-ε-N-biotinyl-l-lysine trideuteromethyl ester ((M + H)+ = 555.0 (555.1 calculated)). Incorporation of the label increases the residue mass of cysteine by 429.2 Da. α-N-iodoacetyl-ε-N-biotinyl- l-lysine methyl ester was prepared similarly using methanol (62 mg; (M + H)+ = 557.8 (558.2 calculated)). Incorporation of the label increases the residue mass of cysteine by 426.2 Da. Membrane pellets were solubilized in 0.5% SDS in 100 m m HEPES, pH 8.5. Disulfide bonds were reduced by the addition of tributyl phosphine to a 1 mm concentration with incubation for 5 min at 90 °C. Cysteine residues were biotinylated using either polyethyleneoxide-iodoacetyl biotin (Pierce) or the ICAT reagents synthesized as above. These sulfhydryl-reactive reagents were added to a concentration of 5 mm and incubated at room temperature in the dark for 1 h. Excess reagents were removed by chloroform/methanol precipitation of the proteins (19Wessel D. Flugge U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.Anal. Biochem. 1984; 138: 141-143Google Scholar), which were resuspended by sonication in digestion buffer (50 mm HEPES, pH 8.5), followed by stepwise addition of SDS and Triton X-100 to concentrations of 0.5 and 1%, respectively. 500 units of PNGase F (New England Biolabs) were added to deglycosylate proteins for 1 h at 37 °C. Modified trypsin (Promega) was added in a 1:50 weight ratio, and digestion was allowed to proceed overnight at 37 °C. Monomeric avidin affinity columns with a 1-ml bed volume (Pierce) were packed according to the manufacturer's instructions. Protein digests were heated to 90 °C for 1 min and treated with AEBSF (Roche Molecular Biochemicals) to inhibit residual trypsin activity and loaded onto the column in phosphate-buffered saline adjusted to pH 6.5. The column was washed with 20 ml of phosphate-buffered saline to remove unlabeled peptides. An extra wash of 3 ml of deionized distilled water removed excess sodium. Biotinylated peptides were then eluted onto a poly-sulfoethyl aspartamide ion exchange column (PolyLC Inc.) with 5 ml of 50 m m trifluoroacetic acid in 25% acetonitrile. The ion exchange column was washed with 750 μl of 0.1% formic acid/25% acetonitrile to remove residual detergent. The peptides were eluted with five 100-μl fractions of 500 mm sodium chloride/0.15% acetic acid, pH 4.1/25% acetonitrile. The fractions were then diluted to 500 μl with 0.1% heptafluorobutyric acid. The fractions were loaded onto a C18 cartridge (1 × 8 mm; Michrom BioResources) and washed with 250 μl of 0.1% heptafluorobutyric acid to remove sodium. The peptides were eluted with 20 μl of 75% acetonitrile/0.1% trifluoroacetic acid. Peptide mixtures (2 μl diluted to 50 μl with 0.1% heptafluorobutyric acid) were loaded onto a 0.25 × 30-mm trapping cartridge packed with Vydac 214MS low trifluoroacetic acid C4 beads. This cartridge was placed in-line with a 0.1 × 100-mm resolving column packed with Vydac 218MS low trifluoroacetic acid C18 beads. The resolving column was constructed using a PicoFritTM (New Objective) fused silica capillary pulled to a 30-μm metal-coated tip, which formed a microelectrospray ionization emitter. Peptides were eluted with a 2-h gradient of 5–80% (v/v) acetonitrile containing 0.1% formic acid/0.005% trifluoroacetic acid at a rate of 0.5 μl/min. Tandem mass spectrometry was performed using an ion trap instrument (LCQ DECA; ThermoFinnigan). For the mass western experiment, three or four selected precursor ions, chosen from the predicted tryptic peptides of the protein of interest, were subjected to collision-induced dissociation (CID) one after the other, cycling repeatedly throughout the LC gradient. Doubly charged ions were assumed for peptides of 8 to 20 residues. A precursor isolation window of 3 Da was used for polyethyleneoxide-iodoacetyl labeled peptides. Heavy and light ICAT-labeled peptides were simultaneously trapped and fragmented by using a 5-Da isolation window. The Sequest data base-searching program was used to generate cross-correlation scores for each CID spectrum versus the predicted peptides. The mass western experimental scheme is illustrated in Fig. 1A. It is similar to the LC-MS-based approaches described above in that relatively crude cellular extracts are labeled with a cysteine-reactive biotinylation reagent and digested without prior separation followed by isolation of the cysteine-containing peptides and analysis by LC-MS/MS. But whereas the object of those experiments was to identify and quantify as many proteins as possible, a more directed approach has been taken to verify the presence of one or a few specific proteins. Given the sequence of a protein, the masses and CID fragmentation patterns of its tryptic peptides are predictable. An LC-MS/MS experiment can therefore be devised that monitors only the predicted tryptic peptides from the protein(s) of interest. The selectivity of tandem mass spectrometry allows all peptides with the wrong precursor masses to be ignored, and the specificity of the fragmentation patterns allows each chosen peptide to be distinguished from all others with the same nominal precursor mass. The number of peptides that can be analyzed in one LC-MS/MS experiment is a function of chromatographic peak widths and the duty cycle of the instrument. With chromatographic peak widths of 20 to 30 s and scan times of 5 s per CID spectrum, three or four ions can be monitored with a strong likelihood of acquiring several spectra for each of the selected ions. Wherever possible, peptides with molecular masses of 1000 to 2500 Da and lacking methionine, tryptophan, or likely sites of post-translational modification are chosen to be assayed. The ICAT methodology was used to achieve quantitative comparisons of samples, but the reagents and scan modes were adapted to optimize sensitivity. Previously reported ICAT experiments used isotope ratios detected in full mass range scans for quantitation, with MS/MS performed in separate scans for protein identification (13Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Google Scholar, 14Griffin T.J. Gygi S.P. Rist B. Aebersold R. Loboda A. Jilkine A. Ens W. Standing K.G. Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer.Anal. Chem. 2001; 73: 978-986Google Scholar). A characteristic of ion trap mass spectrometers is extraordinarily high sensitivity for MS/MS experiments because of the ability to trap and accumulate precursor ions. Under the conditions used in these experiments high quality CID spectra of peptides can be obtained at the 250 attomole level, whereas the limit of detection in full mass MS mode is on the order of 5 to 10 femtomoles. Peptides can therefore be detected by MS/MS that would otherwise be lost in the background noise of full mass range MS. This property of ion traps has been exploited, for example, to obtain MS/MS data on peptides initially detected by matrix-assisted laser desorption-ionization-time-of-flight MS (20Gillece-Castro, B. L., Arnott, D., Henzel, W. J., Bourell, J. H., and Stults, J. T. (1997) Parent Ion Selection for Low Level Ion Trap MS/MS by MALDI-TOF: A Solution to the Lack of Precursor Ion Scans on the Quadrupole Ion Trap. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, California, June 1–5, 1997Google Scholar, 21Zhang X. Herring C.J. Romano P.R. Szczepanowska J. Brzeska H. Hinnebusch A.G. Qin J. Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis.Anal. Chem. 1998; 70: 2050-2059Google Scholar). To take advantage of the sensitivity of the ion trap for MS/MS new ICAT reagents were synthesized that incorporate three (rather than eight) deuterium atoms in the heavy version (Fig. 1B) so that the multiply charged precursor ions of both heavy and light ICAT-labeled peptides are simultaneously trapped and subjected to CID. Fragment ions in the CID spectrum that contain cysteine thus appear as doublets separated by 3 Da allowing both quantitation and identification to be derived from a single scan function performed at maximal sensitivity. The intrinsic sensitivity of capillary HPLC microelectrospray ionization MS and MS/MS on ion trap mass spectrometers is extremely high; efficient sample preparation is therefore a key factor to success. The losses associated with several steps outlined in Fig. 1 were assessed. The initial protein extraction appears robust, as evidenced by minimal residue after solubilization. The extent of labeling with the ICAT reagents is difficult to determine on the membrane preparations themselves, but bovine serum albumin labeled for 30 min followed by addition of excess iodoacetamide revealed no incorporation of the second reagent (data not shown). The removal of excess alkylating reagents after labeling is essential, because they are present in excess over proteins and otherwise dominate the mass spectra. These reagents are not removed by reverse phase, avidin, or size exclusion (nominal 10-kDa cutoff) chromatography. Reaction with tributyl phosphine yields a positively charged molecule, so cation exchange chromatography is only partially effective, although use of this method has been reported (22Ideker T. Thorsson V. Ranish J.A. Christmas R. Buhler J. Eng J.K. Bumgarner R. Goodlett D.R. Aebersold R. Hood L. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network.Science. 2001; 292: 929-934Google Scholar). Chloroform-methanol precipitation, however, effectively removed the reagents. Resolubilization of the precipitated proteins was efficient, with amino acid analysis of the labeled membrane proteins indicating 92% recovery for this procedure. Acid hydrolysis of the labeled proteins cleaves the amide bonds internal to the biotinylation reagents converting each labeled cysteine to carboxymethyl cysteine, which serves as a marker to track the abundance of labeled peptides through the analysis. The use of SDS facilitates solubilization of the membrane proteins but poses a problem for enzymatic digestion. Trypsin activity is almost undetectable at SDS concentrations above 0.25% as measured by a synthetic substrate p-nitroaniline assay; even at 0.05% SDS, 25% of activity is lost. But if a zwitterionic or nonionic detergent like Triton X-100 is first added to an equal or greater concentration than the SDS, trypsin retains its full activity (Fig. 2A). The bands between 10 and 20 kDa observed post-digest are consistent with the presence of trypsin, and the high molecular mass smear is also found in control samples; essentially complete digestion is inferred. Purification of the labeled peptides on the avidin column is another step where sample can be lost. As determined by amino acid analysis, 82% of labeled peptides were recovered when eluted from the column. Of the peptides recovered, over 90% eluted in the second and third fractions collected (1 ml = 1 column volume per fraction), and 99% eluted in the first four fractions. Prostate stem cell antigen was detected in a cell line engineered for its expression. PSCA, a 123-amino acid glycoprotein in the family of glycosylphosphatidylinositol-anchored cell surface antigens, is predominantly expressed in prostate epithelium and is overexpressed in a majority of human prostate cancers (23Reiter R.E. Gu Z. Watabe T. Thomas G. Szigeti K. Davis E. Wahl M. Nisitani S. Yamashiro J. Beau M.M.L. Loda M. Witte O. Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1735-1740Google Scholar). PSCA is a very hydrophobic protein, having a grand average hydropathy score of 0.48, typical of membrane proteins (24Lobry J.R. Gautier C. Hydrophobicity, expressivity and aromaticity are the major trends of amino-acid usage in 999 Escherichia coli chromosome-encoded genes.Nucleic Acids Res. 1994; 22: 3174-3180Google Scholar). Grand average hydropathy scores in several model organisms range from −2.2 (most hydrophilic) to 1.7 (most hydrophobic) (25Wilkins M.R. Gasteiger E. Sanchez J.-C. Bairoch A. Hochstrasser D.F. Two-dimensional gel electrophoresis for proteome projects: the effect of protein hydropathy and copy number.Electrophoresis. 1998; 19: 1501-1505Google Scholar). Despite progress (26Chevallet M. Santoni V. Poinas A. Rouquie D. Fuchs A. Kieffer S. Rossignol M. Lunardi J. Garin J. Rabilloud T. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis.Electrophoresis. 1998; 19: 1901-1909Google Scholar, 27Pasquali C. Fialka I. Huber L.A. Preparative two-dimensional gel electrophoresis of membrane proteins.Electrophoresis. 1997; 18: 2573-2581Google Scholar, 28Molloy M.P. Herbert B.R. Walsh B.J. Tyler M.I. Traini M. Sanchez J.-C. Hochstrasser D.F. Williams K.L. Gooley A.A. Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis.Electrophoresis. 1998; 19: 837-844Google Scholar), only a few proteins with positive grand average hydropathy scores have been identified by 2D electrophoresis (29Molloy M.P. Herbert B.R. Williams K.L. Gooley A.A. Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis.Electrophoresis. 1999; 20: 701-704Google Scholar, 30Molloy M.P. Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients.Anal. Biochem. 2000; 280: 1-10Google Scholar). PC3 cells otherwise lacking PSCA were stably transfected with the PSCA gene. An expression level of ∼200,000 copies per cell as estimated by Scatchard analysis (data not shown) was obtained in the cell line designated clone 11. A subcellular fraction enriched in plasma membrane was prepared from ∼80 million cells by differential sedimentation; one-third of the total PSCA was recovered in this fraction, with the remainder found among the cellular debris pellet as determined by Western blot (data not shown). Proteins in the clone 11 membrane pellet were solubilized, disulfide bonds were reduced, and cysteines were labeled with commercially available polyethyleneoxide-iodoacetyl biotin. Following removal of excess reagents and lipids, proteins were deglycosylated and digested with trypsin. The course of these steps was assessed by SDS-PAGE (Fig. 2B). A Western blot probed with anti-PSCA before deglycosylation (lane 1) and after (lane 2) demonstrates consolidation of a diffuse 25–30-kDa band to a compact band at 14 kDa. Precipitated protein was efficiently recovered (lane 3 versus lane 2). Native PSCA resisted trypsin digestion but was effectively cleaved following deglycosylation (lane 4). A silver-stained gel (Fig. 2A) illustrates the complexity of PC3 membrane fractions (lane 1) and completeness of digestion (lane 2). The presence of PSCA in the clone 11 sample was proven by detection of the peptide GCSLNCVDDSQDYYVGK in the avidin-purified tryptic digest. An aliquot (10%) of the peptide mixture was analyzed by capillary reverse phase LC-microspray ion trap mass spectrometry. The instrument data system was programmed to alternately collect full mass range spectra and product ion spectra of ions with m/z 1347, the anticipated doubly charged ion of the labeled PSCA peptide. Although unnecessary for the mass western experiment, the full mass range spectra were acquired so that the overall complexity of the sample could be judged. The results are diagrammed in Fig. 3. As expected, the full mass range spectra are exceedingly complex (Fig. 3A), with many coeluting peptides at every time point. An extracted ion chromatogram for ions with m/z 1347 is also complex, with over 50 discrete peaks apparent (Fig. 3B). The PSCA peptide was distinguished from all others by its fragmentation pattern. A reconstructed ion chromatogram for ions with m/z 1347 that fragment to form a product with m/z 1189, corresponding to the y10 ion of the PSCA peptide, shows only two peaks, a small one with a retention time of 55 min and a larger peak at 64 min (Fig. 3C). The Sequest algorithm (31Yates III, J.R. Eng J.K. McCormack A.L. Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases.Anal. Chem. 1995; 67: 3202-3210Google Scholar) was used to compare every CID spectrum to the calculated product ions of the PSCA peptide by searching a data base consisting only of the sequence of PSCA. When the cross-correlation score reported by Sequest is plotted for each CID spectrum (Fig. 3D) it is apparent that both major and minor peaks match PSCA. Examination of the complete CID spectra for both peaks revealed them to be qualitatively identical and containing extended series of
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