Abstract
Identities ascribed to the intact protein ions detected in MALDI-MS of whole bacterial cells or from other complex mixtures are often ambiguous. Isolation of candidate proteins can establish that they are of correct molecular mass and sufficiently abundant, but by itself is not definitive. An in vivo labeling strategy replacing methionine with selenomethionine has been employed to deliver an additional constraint for protein identification, i.e., number of methionine residues, derived from the shift in mass of labeled versus unlabeled proteins. By stressing a culture and simultaneously labeling, it was possible to specifically image the cells’ response to the perturbation. Because labeled protein is only synthesized after application of the stress, it provides a means to view dynamic changes in the cellular proteome. These methods have been applied to identify a 15,879 Da protein ion from E. coli that was induced by an antibacterial agent with an unknown mechanism of action as SpY, a stress protein produced abundantly in spheroplasts. It has also allowed us to propose protein identities (and eliminate others from consideration) for many of the ions observed in MALDI (and ESI-MS) whole cell profiling at a specified growth condition.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Cain, T. C.; Lubman, D. M.; Weber, W. J., Jr. Differentiation of Bacteria Using Protein Profiles from Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1994, 8, 1026–1030.
Krishnamurthy, T.; Ross, P. L.; Rajamani, U. Detection of Pathogenic and Non-Pathogenic Bacteria by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 883–888.
Krishnamurthy, T.; Ross, P. L. Rapid Identification of Bacteria by Direct Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Whole Cells. Rapid Commun. Mass Spectrom. 1996, 10, 1992–1996.
Arnold, R. J.; Karty, J. A.; Ellington, A. D.; Reilly, J. P. Monitoring the Growth of a Bacteria Culture by MALDI-MS of Whole Cells. Anal. Chem. 1999, 71, 1990–1996.
Arnold, R. J.; Reilly, J. P. Observation of Escherichia coli Ribosomal Proteins and Their Posttranslational Modifications by Mass Spectrometry. Anal. Biochem. 1999, 269, 105–112.
Arnold, R. J.; Reilly, J. P. Fingerprint Matching of E. coli Strains with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Whole Cells Using a Modified Correlation Approach. Rapid Commun. Mass Spectrom. 1998, 12, 630–636.
Claydon, M. A.; Davey, S. N.; Edwards-Jones, V.; Gordon, D. B. The Rapid Identification of Intact Microorganisms Using Mass Spectrometry. Nature Biotech. 1996, 14, 1584–1586.
Conway, G. C.; Smole, S. C.; Sarracino, D. A.; Arbeit, R. D.; Leopold, P. E. Phyloproteomics: Species Identification of Enterobacteriaceae Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. J. Mol. Microbiol. Biotechnol. 2001, 3, 103–112.
Easterling, M. L.; Colangelo, C. M.; Scott, R. A.; Amster, I. J. Monitoring Protein Expression in Whole Bacterial Cells with MALDI Time-of-Flight Mass Spectrometry. Anal. Chem. 1998, 70, 2704–2709.
Winkler, M. A.; Hickman, R. K.; Golden, A.; Aboleneen, H. Analysis of Recombinant Protein Expression by MALDI-TOF Mass Spectrometry of Bacterial Colonies. BioTechniques 2000, 28, 890–895.
Villanueva, J.; Canals, F.; Querol, E.; Aviles, F. X. Monitoring the Expression and Purification of Recombinant Proteins by MALDI-TOF Mass Spectrometry. Enzyme Microb. Technol. 2001, 29, 99–103.
Liang, X.; Zheng, K.; Qian, M. G.; Lubman, D. M. Determination of Bacterial Protein Profiles by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with High-Performance Liquid Chromatography. Rapid Commun. Mass Spectrom. 1996, 10, 1219–1226.
Jarman, K. H.; Daly, D. S.; Petersen, C. E.; Saenz, A. J.; Valentine, N. B.; Wahl, K. L. Extracting and Visualizing Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectral Fingerprints. Rapid Commun. Mass Spectrom. 1999, 13, 1586–1594.
Bundy, J.; Fenselau, C. Lectin-Based Affinity Capture for MALDI-MS Analysis of Bacteria. Anal. Chem. 1999, 71, 1460–1463.
Madonna, A. J.; Basile, F.; Furlong, E.; Voorhees, K. J. Detection of Bacteria from Biological Mixtures Using Immunomagnetic Separation Combined with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 2001, 15, 1068–1074.
Smole, S. C.; King, L. A.; Leopold, P. E.; Arbeit, R. D. Sample Preparation of Gram-Positive Bacteria for Identification by Matrix Assisted Laser Desorption/Ionization Time-of-Flight. J. Microbiol. Methods 2002, 48, 107–115.
Traldi, P. Qualitative Characterization of Bacterial Strains Employed in the Production of Yogurt by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Mass Spectrom. 1999, 34, 1385–1388.
Dai, Y.; Li, K.; Roser, D. C.; Long, S. R. Detection and Identification of Low-Mass Peptides and Proteins from Solvent Suspensions of Escherichia coli by High Performance Liquid Chromatography Fractionation, and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 1999, 13, 73–78.
Lay, J. O. MALDI-TOF Mass Spectrometry and Bacterial Taxonomy. Trends Anal. Chem. 2000, 19, 507–516.
Lay, J. O., Jr.; Holland, R. D. Rapid Identification of Bacteria Based on Spectral Patterns Using MALDI-TOFMS. Methods Mol. Biol. (Totowa, N. J.) 2000, 146, 461–487.
Demirev, P. A.; Ho, Y.-P.; Ryzhov, V.; Fenselau, C. Microorganism Identification by Mass Spectrometry and Protein Database Searches. Anal. Chem. 1999, 71, 2732–2738.
Ryzhov, V.; Fenselau, C. Characterization of the Protein Subset Desorbed by MALDI from Whole Bacterial Cells. Anal. Chem. 2001, 73, 746–750.
Ryzhov, V.; Hathout, Y.; Fenselau, C. Rapid Characterization of Spores of Bacillus Cereus Group Bacteria by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. Appl. Environ. Microbiol. 2000, 66, 3828–3834.
Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; Gantt, S. L.; Jarman, K. H.; Kingsley, M. T.; Wahl, K. L. Reproducibility of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Replicate Bacterial Culture Analysis. Rapid Commun. Mass Spectrom. 1999, 13, 1580–1585.
Gantt, S. L.; Valentine, N. B.; Saenz, A. J.; Kingsley, M. T.; Wahl, K. L. Use of an Internal Control for Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Analysis of Bacteria. J. Am. Soc. Mass Spectrom. 1999, 10, 1131–1137.
Wilcox, S. K.; Cavey, G. S.; Pearson, J. D. Single Ribosomal Protein Mutations in Antibiotic-Resistant Bacteria Analyzed by Mass Spectrometry. Antimicrob. Agents Chemother. 2001, 45, 3046–3055.
Wang, Z.; Russon, L.; Li, L.; Roser, D. C.; Long, S. R. Investigation of Spectral Reproducibility in Direct Analysis of Bacterial Proteins by MALDI-TOFMS. Rapid Commun. Mass Spectrom. 1998, 12, 456–464.
Haag, A. M.; Taylor, S. N.; Johnston, K. H.; Cole, R. B. Rapid Identification and Speciation of Haemophilus Bacteria by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. J. Mass Spectrom. 1998, 33, 750–756.
Lynn, E. C.; Chung, M.-C.; Tsai, W.-C.; Han, C.-C. Identification of Enterobacteriaceae Bacteria by Direct Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Whole Cells. Rapid Commun. Mass Spectrom. 1999, 13, 2022–2027.
Leenders, F.; Stein, T. H.; Kablitz, B.; Franke, P.; Vater, J. Rapid Typing of Bacillus subtilis Strains by Their Secondary Metabolites Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Intact Cells. Rapid Commun. Mass Spectrom. 1999, 13, 943–949.
Nilsson, C. L. Fingerprinting of Helicobacter pylori Strains by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis. Rapid Commun. Mass Spectrom. 1999, 13, 1067–1071.
Edwards-Jones, V.; Claydon, M. A.; Evason, D. J.; Walker, J.; Fox, A. J.; Gordon, D. B. Rapid Discrimination Between Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus by Intact Cell Mass Spectrometry. J. Med. Microbiol. 2000, 49, 295–300.
Winkler, M. A.; Uher, J.; Cepa, S. Direct Analysis and Identification of Helicobacter and Campylobacter Species by MALDI-TOF Mass Spectrometry. Anal. Chem. 1999, 71, 3416–3419.
Cavalcoli, J. D.; VanBogelen, R. A.; Andrews, P. C.; Moldover, B. Unique Identification of Proteins from Small Genome Organisms: Theoretical Feasibility of High Throughput Proteome Analysis. Electrophoresis 1997, 18, 2703–2708.
Anderson, G.; Bruce, J. E.; Pasa Tolic, L.; Smith, R. D. Proceedings of the 46th Conference of the American Society for Mass Spectrometry; Orlando, FL, 1998, p 1270.
Karty, J. A.; Lato, S.; Reilly, J. P. Detection of the Bacteriological Sex Factor in E. coli by Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1998, 12, 625–629.
Arnold, R. J.; Reilly, J. P. Observation of Tetrahydrofolypolyglutamic Acid in Bacteria Cells by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Anal. Biochem. 2000, 281, 45–54.
Holland, R. D.; Duffy, C. R.; Rafii, F.; Sutherland, J. B.; Heinze, T. M.; Holder, C. L.; Voorhees, K. J.; Jackson, O. L., Jr. Identification of Bacterial Proteins Observed in MALDI TOF Mass Spectra from Whole Cells. Anal. Chem. 1999, 71, 3226–3230.
Loo, R. R.; Cavalcoli, J. D.; VanBogelen, R. A.; Mitchell, C.; Loo, J. A.; Moldover, B.; Andrews, P. C. Virtual 2-D Gel Electrophoresis: Visualization and Analysis of the E. coli Proteome by Mass Spectrometry. Anal. Chem. 2001, 73, 4063–4070.
Bairoch, A. R.; Apweiler, R. A. The SWISS-PROT Protein Sequence Database and Its Supplement Trembl in 2000. Nucleic Acids Res. 2000, 28, 45–48.
Edman, M.; Jarhede, T.; Sjostrom, M.; Wieslander, A. Different Sequence Patterns in Signal Peptides from Mycoplasmas, Other Gram-Positive Bacteria, and Escherichia coli: A Multivariate Data Analysis. Prot. Struct. Funct. Genet. 1999, 35, 195–205.
von Heijne, G. A New Method for Predicting Signal Sequence Cleavage Sites. Nucleic Acids Res. 1986, 14, 4683–4690.
Ben-Bassat, A.; Bauer, K.; Chang, S. Y.; Myambo, K.; Boosman, A.; Chang, S. Processing of the Initiation Methionine from Proteins: Properties of the Escherichia coli Methionine Aminopeptidase and Its Gene Structure. J. Bacteriol. 1987, 169, 751–757.
Boissel, J. P.; Kasper, T. J.; Shah, S. C.; Malone, J. I.; Bunn, H. F. Amino-Terminal Processing of Proteins: Hemoglobin South Florida, a Variant with Retention of Initiator Methionine and Nα-Acetylation. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 8448–8452.
Dalboge, H. S.; Bayne, S.; Pedersen, J. In Vivo Processing of N-Terminal Methionine in Escherichia coli. FEBS Lett. 1990, 266, 1–3.
Hirel, P.-H.; Schmitter, J.-M.; Dessen, P.; Fyat, G.; Blanquet, S. Extent of N-Terminal Methionine Excision from Escherichia coli Proteins Is Governed by the Side Chain Length of the Penultimate Amino Acid. Biochemistry 1989, 86, 8247–8251.
Garrels, J. I.; Futcher, B.; Kobayashi, R.; Latter, G. I.; Schwender, B.; Volpe, T.; Warner, J. R.; McLaughlin, C. S. Protein Identifications for a Saccharomyces cerevisiae Protein Database. Electrophoresis 1994, 15, 1466–1486.
Maillet, I.; Lagniel, G.; Perrot, M.; Boucherie, H.; Labarre, J. Rapid Identification of Yeast Proteins on Two-Dimensional Gels. J. Biol. Chem. 1996, 271, 10263–10270.
Chen, X.; Fei, Z.; Smith, L. M.; Bradbury, E. M.; Majidi, V. Stable-Isotope-Assisted MALDI-TOF Mass Spectrometry for Accurate Determination of Nucleotide Compositions of PCR Products. Anal. Chem. 1999, 71, 3118–3125.
Chen, X.; Smith, L. M.; Bradbury, E. M. Site-Specific Mass Tagging with Stable Isotopes in Proteins for Accurate and Efficient Protein Identification. Anal. Chem. 2000, 72, 1134–1143.
Hunter, T. C.; Yang, L.; Zhu, H.; Majidi, V.; Bradbury, E. M.; Chen, X. Peptide Mass Mapping Constrained with Stable Isotope-Tagged Peptides for Identification of Protein Mixtures. Anal. Chem. 2001, 73, 4891–4902.
Goodlett, D. R.; Bruce, J. E.; Anderson, G. A.; Rist, B.; Pasa-Tolic, L.; Fiehn, O.; Smith, R. D.; Aebersold, R. Protein Identification with a Single Accurate Mass of a Cysteine-Containing Peptide and Constrained Database Searching. Anal. Chem. 2000, 72, 1112–1118.
Veenstra, T. D.; Martinovic, S.; Anderson, G. A.; Pasa-Tolic, L.; Smith, R. D. Proteome Analysis Using Selective Incorporation of Isotopically Labeled Amino Acids. J. Am. Soc. Mass Spectrom. 2000, 11, 78–82.
Blattner, F. R.; Plunkett, G., III; Bloch, C. A.; Perna, N. T.; Burland, V.; Riley, M.; Collado-Vides, J.; Glasner, J. D.; Rode, C. K.; Mayhew, G. F.; Gregor, J.; Davis, N. W.; Kirkpatrick, H. A.; Goeden, M. A.; Rose, D. J.; Mau, B.; Shao, Y. The Complete Genome Sequence of Escherichia coli K-12. Science 1997, 277, 1453–1474.
Itoh, T.; Okayama, T.; Hashimoto, H.; Takeda, J.-I.; Davis, R. W.; Mori, H.; Gojobori, T. A Low Rate of Nucleotide Changes in Escherichia coli K-12 Estimated from a Comparison of the Genome Sequences between Two Different Substrains. FEBS Lett. 1999, 450, 72–76.
VanBogelen, R. A.; Neidhardt, F. C. Preparation of Escherichia coli Samples for Two-Dimensional Gel Analysis. Link, A., Ed. In Methods in Molecular Biology: 2-D Proteome Analysis Protocols, Vol. CXII. Humana Press: Totowa, 1998; pp 21–29.
VanBogelen, R. A. Generating a Bacterial Genome Inventory. Link, A., Ed. In Methods in Molecular Biology: 2-D Proteome Analysis Protocols, Vol. CXII. Humana Press: Totowa, 1998; pp 423–429.
Benjamin, D. R.; Robinson, C. V.; Hendrick, J. P.; Hartl, F. U.; Dobson, C. M. Mass Spectrometry of Ribosomes and Ribosomal Subunits. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7391–7395.
Gygi, 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, 994–999.
Jensen, O. N.; Larsen, M. R.; Roepstorff, P. Mass Spectrometric Identification and Microcharacterization of Proteins from Electrophoretic Gels: Strategies and Applications. Prot. Struct. Funct. Genet. Suppl. 1998, 2, 74–89.
Sechi, S.; Chait, B. T. Modification of Cysteine Residues by Alkylation. A Tool in Peptide Mapping and Protein Identification. Anal. Chem. 1998, 70, 5150–5158.
James, P.; Quadroni, M.; Carafoli, E.; Gonnet, G. Protein Identification in DNA Databases by Peptide Mass Fingerprinting. Protein Science 1994, 3, 1347–1350.
Chaurand, P.; Luetzenkirchen, F.; Spengler, B. Peptide and Protein Identification by Matrix-Assisted Laser Desorption Ionization (MALDI and MALDI-Post-Source Decay Time-of-Flight Mass Spectrometry). J. Am. Soc. Mass Spectrom. 1999, 10, 91–103.
Hale, J. E.; Butler, J. P.; Knierman, M. D.; Becker, G. W. Increased Sensitivity of Tryptic Peptide Detection by MALDI-TOF Mass Spectrometry Is Achieved by Conversion of Lysine to Homoarginine. Anal. Biochem. 2000, 287, 110–117.
Beardsley, R. L.; Karty, J. A.; Reilly, J. P. Enhancing the Intensities of Lysine-Terminated Tryptic Peptide Ions in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 2147–2153.
Dayhoff, M. O. Atlas of Protein Sequence and Structure, Suppl. 3; National BioMedical Research Foundation: Washington, 1976.
Eaton, R. P.; Pommer, I. Incorporation of Selenium-75-Labeled Selenomethionine into Human Apoproteins. I. Characterization of Specificity in Very-Low-Density and Low-Density Lipoproteins. Diabetes 1976, 25, 32–43.
Easty, D. J.; Patel, K.; Dover, R.; Evans, D. J.; Dunn, M. J. A Comparative Study of Protein Synthesis by Keratinocytes and Fibroblasts in Vitro Using Two-Dimensional Gel Electrophoresis and Dual Isotope Autoradiography. Electrophoresis (Weinheim, Germany) 1988, 9, 227–231.
Hagenmaier, S.; Stierhof, Y.-D.; Henning, U. A New Periplasmic Protein of Escherichia coli Which Is Synthesized in Spheroplasts but Not in Intact Cells. J. Bacteriol. 1997, 179, 2073–2076.
Pasa-Tolic, L.; Jensen, P. K.; Anderson, G. A.; Lipton, M. S.; Peden, K. K.; Martinovic, S.; Tolic, N.; Bruce, J. E.; Smith, R. D. High Throughput Proteome-Wide Precision Measurements of Protein Expression Using Mass Spectrometry. J. Am. Chem. Soc. 1999, 121, 7949–7950.
Griffin, 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, 978–986.
Coch, E. H.; Greene, R. C. Utilization of Selenomethionine by Escherichia coli. Biochim. Biophys. Acta 1971, 230, 223–236.
Pedersen, S.; Bloch, P. L.; Reeh, S.; Neidhardt, F. C. Patterns of Protein Synthesis in E. coli: A Catalog of the Amount of 140 Individual Proteins at Different Growth Rates. Cell 1978, 14, 179–190.
Wilkins, M. R.; Gasteiger, E.; Bairoch, A.; Sanchez, J.-C.; Williams, K. L.; Appel, R. D.; Hochstrasser, D. F. Protein Identification and Analysis Tools in the Expasy Server. In 2-D Proteome Analysis Protocols; Link, A. J., Ed.; Humana Press: Totowa, 1998; pp 531–552.
Link, A. J.; Robison, K.; Church, G. M. Comparing the Predicted and Observed Properties of Proteins Encoded in the Genome of Escherichia coli K-12. Electrophoresis 1997, 18, 1259–1313.
Pfu Methionine Aminopeptidase. Otsu, Shiga, Japan. Takara Biotechnology (Dalian) Co., Ltd., 2001.
Ge, Y.; Lawhorn, B. G.; ElNaggar, M.; Strauss, E.; Park, J.-H.; Begley, T. P.; McLafferty, F. W. Top Down Characterization of Larger Proteins (45 kDa) by Electron Capture Dissociation Mass Spectrometry. J. Am. Chem. Soc. 2002, 124, 672–678.
Meng, F.; Cargile, B. J.; Miller, L. M.; Forbes, A. J.; Johnson, J. R.; Kelleher, N. L. Informatics and Multiplexing of Intact Protein Identification in Bacteria and the Archaea. Nat. Biotechnol. 2001, 19, 952–957.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Loo, R.R.O., Loo, J.A., Du, P. et al. In vivo labeling: A glimpse of the dynamic proteome and additional constraints for protein identification. J Am Soc Mass Spectrom 13, 804–812 (2002). https://doi.org/10.1016/S1044-0305(02)00408-7
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1016/S1044-0305(02)00408-7