Selective Detection of Membrane Proteins Without Antibodies

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. 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 with a J. J. I. and Scholar). the ErbB2 experiment the cell was at 1000 for to intact cells and The was at for to a crude membrane the experiment the was a and in a at for The membrane was and for with each of the to proteins A. of plasma identification of Biol. Scholar), at and with each by at for were by was in a was in and in a to and more from The was used without The was in and and with After the reaction mixture was a in of and using a gradient were analyzed mass spectrometry and and to of the the mass of by was using of the the mass of by Membrane were in in were by the of to a with for at were using or the ICAT reagents as These reagents were to a of and at in the for reagents were by of the proteins D. A method for the of protein in in the presence of and Biochem. Scholar), which were by in digestion by of and to of and respectively. of were to proteins for at was in a and digestion was to at affinity with a were to the digests were to for and with to and the in to The was with of to of of peptides were a ion with of in The ion was with of to The peptides were with of The were to with The were a and with of to The peptides were with of mixtures to with were a with This was with a resolving with The resolving was using a to a which a were with a gradient of at a of Tandem mass spectrometry was performed using an ion trap For the mass western experiment, or selected from the tryptic peptides of the protein of were subjected to collision-induced dissociation the the were for peptides of to A isolation of was used for labeled and light peptides were simultaneously trapped and fragmented by using a isolation The data was used to for each spectrum the The mass western is in is similar to the approaches described in that crude cellular extracts are labeled with a biotinylation and without separation by isolation of the peptides and analysis by the of experiments was to identify and as many proteins as a more has been to the presence of or a few specific proteins. the sequence of a protein, the and patterns of tryptic peptides are experiment can be that only the tryptic peptides from the of interest. The of tandem mass spectrometry allows peptides with the to be and the of the patterns allows each peptide to be from with the mass. The of peptides that can be analyzed in experiment is a function of and the of the of to and of or can be with a of several spectra for each of the selected peptides with of 1000 to 2500 and or of post-translational are to be The ICAT methodology was used to of but the reagents and were to ICAT experiments used detected in mass for with performed in 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 of ion trap mass is extraordinarily high sensitivity for experiments because of the ability to trap and the used in these experiments high spectra of peptides can be at the the of detection in mass MS is on the of to can be detected by that would otherwise be in the of mass This of ion has been for to data on peptides detected by MS B. W. J. and J. for by A to the of on the of the on Mass and Scholar, J. J. of in proteins by Chem. 1998; Scholar). of the sensitivity of the ion trap for ICAT reagents were that in the heavy that the of heavy and light peptides are simultaneously trapped and subjected to in the spectrum that as by quantitation and identification to be from a single function performed at The sensitivity of MS and on ion trap mass is extremely sample is a to The with several in were The initial protein as by The of labeling with the ICAT reagents is difficult to on the membrane preparations but labeled for by of of the not The of reagents labeling is because they are in proteins and otherwise the mass These reagents are not by or chromatography. with a chromatography is only use of this method has been R. J. Eng R. D.R. Aebersold R. L. genomic and proteomic of a 2001; Scholar). however, the of the proteins was with analysis of the labeled membrane proteins for this of the labeled proteins the to the biotinylation reagents each labeled to which as a to the abundance of labeled peptides the The use of of the membrane proteins but a for is at as by a even at of is a or is first to an or the The between and are with the presence of and the high mass is in digestion is of the labeled peptides on the is where sample can be As by of labeled peptides were from the the peptides in the and and in the first stem cell antigen was detected in a cell line for PSCA, a in the of cell surface is expressed in prostate and is overexpressed in a of human prostate G. Davis E. M. S. J. M. stem cell a cell surface overexpressed in prostate Natl. Acad. Sci. U. S. A. 1998; Scholar). PSCA is a protein, a of of membrane proteins J.R. and are the of in Scholar). in several from to E. A. electrophoresis for proteome the of protein and 1998; 19: Scholar). M. A. D. A. S. M. J. J. the analysis of membrane proteins by two-dimensional 1998; 19: Scholar, I. two-dimensional electrophoresis of membrane 1997; 18: Scholar, M.P. B.R. M. of membrane proteins by differential for separation using two-dimensional 1998; 19: Scholar), only a few proteins with have been identified by 2D electrophoresis M.P. B.R. of proteins with to two-dimensional 1999; Scholar, M.P. electrophoresis of membrane proteins using Biochem. 2000; Scholar). PC3 cells otherwise PSCA were transfected with the PSCA expression of cell as by analysis not was in the cell line clone A in plasma membrane was from million cells by differential of the total PSCA was in this with the the cellular as by Western blot not Proteins in the clone membrane were were and were labeled with of reagents and proteins were and with The of these was by A Western blot with and of a to a at protein was PSCA digestion but was A the of PC3 membrane and of digestion The presence of PSCA in the clone sample was by detection of the peptide in the tryptic of the peptide mixture was analyzed by ion trap mass spectrometry. The data was to mass spectra and ion spectra of with the anticipated ion of the labeled PSCA Although for the mass western experiment, the mass spectra were that the of the sample be The results are in As the mass spectra are complex with many peptides at ion for with is with peaks The PSCA peptide was from by A ion for with that to a with corresponding to the ion of the PSCA only two a with a of and a at The III, J.R. Eng tandem mass spectra of and peptides to in Chem. Scholar) was used to spectrum to the of the PSCA peptide by searching a data base only of the sequence of the by is for each spectrum it is that and peaks of the spectra for peaks to be and extended of for the PSCA peptide corresponds to the The for the of the peptide is but may be because of in this and quantitation of proteins by ICAT methodology was by 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) by comparison of peptide MS but of proteins from data samples were labeled with the ICAT reagents and in of and digested, and analyzed by The spectrum of the labeled tryptic peptide is in and is for each Each of the with the labeled and these peaks as from which data can be and from the ion from each separation was between and and were the a with peptides to This is because a in between heavy and light would have a on et al. R. C.S. S. of labeled peptides in Chem. 2001; 73: Scholar) have and with the that and peptides were to in with for and peptides R. C.S. S. of labeled peptides in Chem. 2001; 73: Scholar). of for of would go in given the of the ion trap quantitation that the light and heavy of a peptide are trapped and fragmented in the ion trap mass the isolation can at the of in A of was to be sufficient for two by the between of peptides that a peptide more the ion is that a with should be or peptide should be Each ion in a spectrum that can be used to relative these are to a more the the peptides and are for the first and The relative are and respectively. More be by the results from multiple peptides from the protein, but even the single peptide for most biological experiments. is that the of the labeled are for of The ion trap mass at the patterns of are A more is of the of a ion with the of the corresponding the of these The abundance of is such that the is of the in peptides and to at This can be the is more the or the two peaks are of similar the is compared with the mass can be used for because they or a can be based on the is to such as is more in sample A sample would to the more sample with the heavy ICAT The receptor tyrosine kinase ErbB2 is overexpressed in of breast W.J. A. breast of and with amplification of the Scholar). A antibody ErbB2 has an for breast as a single and in with M.A. D. S. L. J.W. S. G. of the and of antibody in have breast that has for 1999; 17: Scholar). levels of ErbB2 have been in a of breast tumor cell lines MCF-7 and MCF-7 cells cell expression SK-BR-3 cells can as many as million on R.W. M.D. P. of on and malignant human breast and 1999; 18: Scholar). of the cells used for experiment an ErbB2 expression of between and by Western blot not The of the mass western experiment was performed to the relative abundance of ErbB2 in these cell Membrane proteins from million MCF-7 cells were labeled with the ICAT and proteins from the of SK-BR-3 cells were labeled with the ICAT Because of the expression levels as protein as by from the MCF-7 membrane was used as from the SK-BR-3 The samples were and with and the peptides were and relative of ErbB2 was by detection of the tryptic peptide a on were subjected to that the of the and peptide were trapped and fragmented A a data base of only the ErbB2 sequence was out for a of ICAT or A of for each spectrum ErbB2 only high with a of separation of the heavy and light labeled was The spectra this were and to the for the ErbB2 tryptic A of this spectrum a data base of human proteins ErbB2 as the of the that the ICAT of peaks by mass (e.g. and in and The relative abundances of the labeled by the an ErbB2 expression of between MCF-7 and SK-BR-3 This with the Western blot The mass western experiment described the use of proteomic techniques for protein This is in to most proteomic to which have used or two-dimensional electrophoresis, multidimensional protein or ICAT LC-MS for the of protein expression data 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, R. J. Eng R. D.R. Aebersold R. L. genomic and proteomic of a 2001; Scholar, and analysis of proteins expressed by and human application of mass spectrometry to Scholar, M. P. L. of proteins. 2001; Scholar, D. Stults J.T. to proteome analysis: identification of proteins with Biochem. 1998; 258: Scholar, analysis of the human cell line of a membrane protein 2000; 21: Scholar, D. Henzel W. S. S. Stults J. M. L. A two-dimensional protein of 1999; Scholar, M.P. A. Y. The yeast and Biol. 2000; for As with Western blots, the mass western technique can detect a specific protein from a complex using an antibody for the of a detection of a peptide is by mass spectrometry. The of the of a peptide is such that results are the peptide for detection is to the protein of a that can be by a of the peptide W. W. Biol. Scholar). as proteins can be with several antibodies, peptides from multiple proteins can be detected in sufficient sample is This experiment from Western in that of the of a protein is because electrophoresis is not of this experiment compared with techniques such as 2D electrophoresis is proteins on the of expressed sequence or genomic DNA should be the gene sequence is and protein that can be with and is potentially These (e.g. proteins such as proteins of proteins with mass and proteins do not tryptic such must be (e.g. and J. A. M. A. M. F. for and analysis in based on Chromatogr. B. 2000; Scholar). the sensitivity of ion trap the detection and of this technique are by sample because peptides to the in the abundance ion may not be a on the abundance ErbB2 expressed at cell was and amounts are with current but many proteins are at numbers of cell or of such proteins require of by of peptide (e.g. J. A. M. A. M. F. for and analysis in based on Chromatogr. B. 2000; Scholar) or tandem 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, 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). We and in the of J. Henzel for the analysis and for the PC3 clone cell Aebersold on the purification of

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.