Hanjo Lim, Aventis Pharmaceuticals Inc., Bridgewater, New Jersey, USA
John R Yates, The Scripps Research Institute, La Jolla, California, USA
Published online: October 2002
DOI: 10.1038/npg.els.0003110
Matrix-assisted laser desorption/ionization mass spectrometry and electrospray tandem mass spectrometry, coupled with liquid chromatography, enable high-throughput protein identification and further characterization, such as post-translational modification, of proteins, after initial separation by two-dimensional gel electrophoresis.
Keywords: proteomics; two-dimensional gel electrophoresis; mass spectrometry; posttranslational modification
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Figure 1. Strategy for high-throughput protein identification from two-dimensional gels. Each two-dimensional gel electrophoresis (2-DE) protein spot is excised and in-gel digested with trypsin. The resulting tryptic digests are initially screened by a combination of matrix-assisted laser desorption/ionization (MALDI) and high-resolution time-of-flight (TOF) mass spectrometry (MS) for protein identification. Peptide mass mapping is sufficient to identify the majority of spots. The technique matches the experimental peptide mass to charge (m/z) values with theoretical m/z values generated from database protein sequences. Samples that cannot be identified by means of mass mapping are subjected to capillary liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS). Each peptide's tandem mass spectrum is automatically compared with theoretically predicted tandem mass spectra by the SEQUEST database search algorithm, which then identifies the proteins comprising the sample. MW, molecular weight.
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| References | |
| Eng JK, McCormack AL and Yates JR III (1994) An approach to correlate mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry 5: 976–989. | |
| Huddleston MJ, Annan RS, Bean MF and Carr SA (1993) Selective detection of phosphopeptides in complex mixtures by electrospray liquid chromatography/mass spectrometry. Journal of the American Society of Mass Spectrometry 4: 710–717. | |
| proceedings Hunt DF, Yates JR III, Shabanowitz J, Winston S and Hauer CR (1986) Protein sequencing by tandem mass spectrometry. Proceedings of the National Academy of Sciences of the USA 83: 6233–6238. | |
| Hunt DF, Henderson RA, Shabanowitz J et al. (1992) Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 259: 1261–1263. | |
| Link AJ, Hays LG, Carmack EB and Yates JR III (1997) Identifying the major proteome components of Haemophilus influenzae type-strain NCTC 8143. Electrophoresis 18: 1314–1334. | |
| Link AJ, Eng J, Schieltz DM et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nature Biotechnology 17: 676–682. | |
| proceedings Mortz E, O'Connor PB, Roepstorff P et al. (1996) Sequence tag identification of intact proteins by matching tandem mass spectral data against sequence data bases. Proceedings of the National Academy of Sciences of the USA 93: 8264–8267. | |
| Pappin DJC, Hojrup P and Bleasby AJ (1993) Rapid identification of proteins by peptide-mass fingerprinting. Current Biology 3: 327–332. | |
| Wu X, Ranganathan V, Seisman DS et al. (2000) ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response. Nature 405: 477–482. | |
| Further Reading | |
| Blackstock WP and Weir MP (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends in Biotechnology 17: 121–127. | |
| book Burlingame AL, Carr SA and Baldwin MA (2000) Mass Spectrometry in Biology and Medicine. Totowa, NJ: Humana Press. | |
| book Wilkins MR, Willams KL, Appel RD and Hochstrasser DF (1997) Proteome Research: New Frontiers in Functional Genomics. Berlin: Springer. | |
| Yates JR III (1998) Mass spectrometry and the age of the proteome. Journal of Mass Spectrometry 33: 1–19. | |
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