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Microbial Proteomics Using Mass Spectrometry

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Microbial Systems Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 881))

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Abstract

Proteomic analyses involve a series of intricate, interdependent steps involving approaches and technical issues that must be fully coordinated to obtain the optimal amount of required information about the test subject. Fortunately, many of these steps are common to most test subjects, requiring only modifications to or, in some cases, substitution of some of the steps to ensure they are relevant to the desired objective of a study. This fortunate occurrence creates an essential core of proteomic approaches and techniques that are consistently available for most studies, regardless of test subject. In this chapter, an overview of some of these core approaches, techniques, and mass spectrometric instrumentation is given, while indicating how such steps are useful for and applied to bacterial investigations. To exemplify how such proteomic concepts and techniques are applicable to bacterial investigations, a practical, quantitative method useful for bacterial proteomic analysis is presented with a discussion of possibilities, pitfalls, and some emerging technology to provide a compilation of information from the diverse literature that is intermingled with experimental experience.

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References

  1. Yarmush ML, Jayaraman A (2002) Advances in proteomic technologies. Annu Rev Biomed Eng 4:349–373

    Article  PubMed  CAS  Google Scholar 

  2. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O’Shea EK, Weissman JS (2003) Global analysis of protein expression in yeast. Nature 425:737–841

    Article  PubMed  CAS  Google Scholar 

  3. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC et al (2001) Initial sequencing and analysis of the human genome. Nature 409:860–921

    Article  PubMed  CAS  Google Scholar 

  4. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ et al (2001) The sequence of the human genome. Science 291:1304–1351

    Article  PubMed  CAS  Google Scholar 

  5. Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467–470

    Article  PubMed  CAS  Google Scholar 

  6. Bowtell DD (1999) Options available—from start to finish—for obtaining expression data by microarray. Nat Genet 21:25–32

    Article  PubMed  CAS  Google Scholar 

  7. Cheung VG, Morley M, Aguilar F, Massimi A, Kucherlapati R, Childs G (1999) Making and reading microarrays. Nat Genet 21:15–19

    Article  PubMed  CAS  Google Scholar 

  8. Duggan DJ, Bittner M, Chen Y, Meltzer P, Trent JM (1999) Expression profiling using cDNA microarrays. Nat Genet 21:10–14

    Article  PubMed  CAS  Google Scholar 

  9. Anderson L, Seilhamer J (1997) A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18:533–537

    Article  PubMed  CAS  Google Scholar 

  10. Lu P, Vogel C, Wang R, Yao X, Marcotte EM (2007) Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation. Nat Biotechnol 25:117–124

    Article  PubMed  CAS  Google Scholar 

  11. Schrimpf SP, Weiss M, Reiter L, Ahrens CH, Jovanovic M, Malmstrom J, Mohanty S, Lercher MJ, Hunziker PE, Aebersold R, von Mering C, Hengartner MO (2009) Comparative functional analysis of the Caenorhabditis elegans and Drosophila melanogaster proteomes. PLoS Biol 7:e48

    Article  PubMed  Google Scholar 

  12. Wilkins MR, Pasquali C, Appel RD, Ou K, Golaz O, Sanchez JC et al (1996) From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology 14:61–65

    Article  PubMed  CAS  Google Scholar 

  13. Lane CS (2005) Mass spectrometry-based proteomics in the life sciences. Cell Mol Life Sci 62:848–869

    Article  PubMed  CAS  Google Scholar 

  14. Cutler P (2003) Protein arrays: the current state-of-the-art. Proteomics 3:3–18

    Article  PubMed  CAS  Google Scholar 

  15. Melton L (2004) Proteomics in multiplex. Nature 429:101–107

    Article  PubMed  Google Scholar 

  16. Domon B, Aebersol R (2006) Mass spectrometry and protein analysis. Science 312:212–217

    Article  PubMed  CAS  Google Scholar 

  17. Fenn J (2002) Electrospray ionization mass spectrometry: how it all began. J Biomol Tech 13:101–118

    PubMed  Google Scholar 

  18. Kara M, Kruger R (2003) Ion formation in MALDI: the cluster ionization mechanism. Chem Rev 103:427–440

    Article  Google Scholar 

  19. Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Hum Genet 26:231–243

    CAS  Google Scholar 

  20. O’Farrell PH (1975) High resolution two dimensional electrophoresis of proteins. J Biol Chem 25:4007–4021

    Google Scholar 

  21. van den Bergh G, Arckens L (2005) Recent advances in 2D electrophoresis: an array of possibilities. Expert Rev Proteomics 2:243–2520

    Article  PubMed  Google Scholar 

  22. Breggen E, Chernokalskaya E, Steinberg TH (2000) Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using luminescent ruthenium complex. Electrophoresis 21:2509–2521

    Article  Google Scholar 

  23. Miller I, Crawford J, Gianazza E (2006) Protein stains for proteomic application: which, when, why? Proteomics 6:5385–5408

    Article  PubMed  CAS  Google Scholar 

  24. Westermeier R, Mmarouga R (2005) Protein detection methods in proteomics research. Biosci Rep 25:19–32

    Article  PubMed  CAS  Google Scholar 

  25. Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077

    Article  PubMed  CAS  Google Scholar 

  26. Wolters DA, Washburn MP, Yates JR III (2001) An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 73:5683–5690

    Article  PubMed  CAS  Google Scholar 

  27. Kline KG, Wu CC (2009) MudPIT analysis: application to human heart tissue. Methods Mol Biol 528:281–293

    Article  PubMed  CAS  Google Scholar 

  28. Issaq HJ, Chan GC, Janini GM, Conrads TP, Veenstra TD (2005) Multidimensional separation of peptides for effective proteomic analysis. J Chromatogr B 817:35–47

    Article  CAS  Google Scholar 

  29. Condina MR, Klingler-Hoffmann M, Hoffmann P (2010) Tyrosine phosphorylation enrichment and subsequent analysis by MALDI-TOF/TOF MS/MS and LC-ESI-IT-MS/MS. Curr Protoc Protein Sci. Chapter 13. doi: 10.1002/0471140864.ps1311supplement62

    Google Scholar 

  30. Kebarle P, Verkerk UH (2009) Electrospray: from ions in solution to ions in the gas phase, what we know now. Mass Spectrom Rev 28:898–917

    Article  PubMed  CAS  Google Scholar 

  31. Hagen JJ, Monnig CA (1994) Method for estimating molecular mass from electrospray spectra. Anal Chem 66:1877–1883

    Article  CAS  Google Scholar 

  32. McLuckey SA, Mentinova M (2011) Ion/neutral, ion/electron, ion/photon, and ion/ion interactions in tandem mass spectrometry: do we need them all? Are they enough? J Am Soc Mass Spectrom 22:3–12

    Article  PubMed  CAS  Google Scholar 

  33. Schevchenko A, Chernushevich I, Wilm M, Mann M (2000) De novo sequencing by nanoelectrospray tandem mass spectrometry using triple quadrupole and quadrupole/time-of-flight instruments. Methods Mol Biol 146:1–16

    Google Scholar 

  34. Knochenmuss R (2006) Ion formation mechanisms in UV-MALDI. Analyst 131:966–986

    Article  PubMed  CAS  Google Scholar 

  35. Silvertand LH, Torano JS, de Jong GJ, van Bennekom WP (2008) Improved repeatability and matrix-assisted desorption/ionization—time of flight mass spectrometry compatibility in capillary isoelectric focusing. Electrophoresis 29:1985–1996

    Article  PubMed  CAS  Google Scholar 

  36. Zheng J, Li N, Ridyard M, Dai H, Robbins SM, Li L (2005) Simple and robust two-layer matrix/sample preparation method for MALDI MS/MS analysis of peptides. J Proteome Res 4:1709–1716

    Article  PubMed  CAS  Google Scholar 

  37. Bornsen KO (2000) Influence of salts, buffers, detergents, solvents, and matrices on MALDI-MS protein analysis in complex mixtures. Methods Mol Biol 146:387–404

    PubMed  CAS  Google Scholar 

  38. Chen YC, Shiea J, Sunner J (1998) Thin-layer chromatography-mass spectrometry using activated carbon, surface-assisted laser desorption/ionization. J Chromatogr A 826:77–86

    Article  PubMed  CAS  Google Scholar 

  39. Shen Z, Thomas JJ, Averbuj C, Broo KM, Engelhard M, Crowell JE, Finn MG, Siuzdak G (2001) Porous silicon as a versatile platform for laser desorption/ionization mass spectrometry. Anal Chem 73:612–619

    Article  PubMed  CAS  Google Scholar 

  40. Kaufmann R (1995) Matrix-assisted laser desorption ionization (MALDI) mass spectrometry: a novel analytical tool in molecular biology and biotechnology. J Biotechnol 41:155–175

    Article  PubMed  CAS  Google Scholar 

  41. Jonsher KR, Yates JR III (1997) The quadrupole ion trap mass spectrometer—a small solution to a big challenge. Anal Biochem 1:1–15

    Article  Google Scholar 

  42. Hopfgartner G, Varesio E, Tschappat V, Grivet C, Bourgogne E, Leuthold LA (2004) Triple quadrupole linear ion trap mass spectrometer for the analysis of small molecules and macromolecules. J Mass Spectrom 39:845–855

    Article  PubMed  CAS  Google Scholar 

  43. Marshall AG, Hendrickson CL, Jackson GS (1998) Fourier transform ion cyclotron resonance mass spectrometry: a primer. Mass Spectrom Rev 17:1–35

    Article  PubMed  CAS  Google Scholar 

  44. Hu Q, Noll RJ, Li H, Makarov A, Hardman M, Graham Cooks R (2005) The orbitrap: a new mass spectrometer. J Mass Spectrom 40:430–443

    Article  PubMed  CAS  Google Scholar 

  45. Lundgren DH, Martinez H, Wright ME, Han DK (2009) Protein identification using Sorcerer 2 and SEQUEST. Curr Protoc Bioinformatics Chapter 13:Unit 13.3

    Google Scholar 

  46. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567

    Article  PubMed  CAS  Google Scholar 

  47. Brosch M, Swamy S, Hubbard T, Choudhary J (2008) Comparison of Mascot and X!Tandem performance for low and high accuracy mass spectrometry and the development of an adjusted Mascot threshold. Mol Cell Proteomics 7:962–970

    Article  PubMed  CAS  Google Scholar 

  48. Elias JE, Gygi SP (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4:207–214

    Article  PubMed  CAS  Google Scholar 

  49. Higdon R, Hogan JM, Ban Belle G, Kolker E (2005) Randomized sequence databases for tandem mass spectrometry peptide and protein identification. OMICS 9:364–379

    Article  PubMed  CAS  Google Scholar 

  50. Karimpour-Fard A, Leach SM, Gill RT, Hunter LE (2008) Predicting protein linkages in bacteria: which method is best depends on task. BMC Bioinformatics 9:397–418

    Article  PubMed  Google Scholar 

  51. du Plessis L, Skunca N, Dessimoz C (2011) The what, where, how, and why of gene ontology—a primer for bioinformaticians. Brief Bioinformatics 12:723–735. doi:10.1093/bib/bbr002

    Article  PubMed  Google Scholar 

  52. Ganter B, Giroux CN (2008) Emerging applications of network and pathway analysis in drug discovery and development. Curr Opin Drug Discov Devel 11:86–94

    PubMed  CAS  Google Scholar 

  53. Wu CC, MacCoss MJ (2002) Shotgun proteomics: tools for the analysis of complex biological systems. Curr Opin Mol Ther 4:242–250

    PubMed  CAS  Google Scholar 

  54. Bogdanov B, Smith RD (2005) Proteomics by FTICR mass spectrometry: top down and bottom up. Mass Spectrom Rev 24:168–200

    Article  PubMed  CAS  Google Scholar 

  55. Waanders LF, Hanke S, Mann M (2007) Top-down quantitation and characterization of SILAC-labeled proteins. J Am Soc Mass Spectrom 18:2058–2064

    Article  PubMed  CAS  Google Scholar 

  56. Patton WF (2002) Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci 771:3–31

    Article  PubMed  CAS  Google Scholar 

  57. Aggrawal K, Choe LH, Lee KH (2006) Shotgun proteomics using the iTRAQ isobaric tags. Brief Funct Genomic Proteomic 5:112–120

    Article  Google Scholar 

  58. Brewis IA, Brennan P (2010) Proteomics technologies for the global identification and quantification of proteins. Adv Protein Chem Struct Biol 80:1–44

    Article  PubMed  CAS  Google Scholar 

  59. de Souza G, Wiker HG (2009) The impact of proteomic advances on bacterial gene annotation. Curr Proteomics 6:84–92

    Google Scholar 

  60. Cash P (2003) Proteomics of bacterial pathogens. Adv Biochem Eng Biotechnol 83:93–115

    PubMed  CAS  Google Scholar 

  61. Collier RJ, Young JA (2003) Anthrax toxin. Annu Rev Cell Dev Biol 19:45–70

    Article  PubMed  CAS  Google Scholar 

  62. Wang J, Ying T, Wang H, Shi Z, Li M, He K, Feng E, Wang J, Yuan J, Li T, Wei K, Su G, Zhu H, Zhang X, Huang P, Huang L (2005) 2-D reference map of Bacillus anthracis vaccine strain A16R proteins. Proteomics 5:4488–4495

    Article  PubMed  CAS  Google Scholar 

  63. Francis AW, Ruggerio CE, Koppisch AT, Dong J, Song J, Brettin T, Iyer S (2005) Proteomic analysis of Bacillus anthracis Sterne vegetative cell. Biochim Biophys Acta 1748:191–200

    Article  PubMed  CAS  Google Scholar 

  64. Antelmann H, Williams RC, Miethke M, Wipat A, Albrecht D, Harwood CR, Hecker M (2005) The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2. Proteomics 5:3684–3695

    Article  PubMed  CAS  Google Scholar 

  65. Gohar M, Bilois N, Graveline R, Garreau C, Sanchis V, Lereclus D (2005) A comparative study of B. cereus, B. thuringiensis, and B. anthracis extracellular proteomes. Proteomics 5:3696–3711

    Article  PubMed  CAS  Google Scholar 

  66. Waltz A, Mujer C, Connolly JP, Alefantis T, Chafein R, Dake C, Whittingtion J, Kumar SP, Khan AS, DelVecchio VG (2007) Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins. Proteome Sci 5:11

    Article  Google Scholar 

  67. Chitlaru T, Shafferman A (2009) Proteomic studies of Bacillus anthracis. Future Microbiol 4:983–998

    Article  PubMed  CAS  Google Scholar 

  68. Liu H, Bergman NH, Thomason B, Shallom S, Hazen A, Crossno J, Rasko DA, Ravel J, Read TD, Peterson SN, Yates J III (2004) Formation and composition of the Bacillus anthracis endospore. J Bacteriol 186:164–178

    Article  PubMed  CAS  Google Scholar 

  69. Li Q, Lostumbo G (2010) Proteomic analyses of a variety of intracellular bacterial species infecting different host cell lines. Curr Proteomics 7:222–232

    Article  CAS  Google Scholar 

  70. Pechous RD, McCarthy TR, Zahrt TC (2009) Working toward the future: insights into Francisella tularensis pathogenesis and vaccine development. Microbiol Mol Biol Rev 73:684–711

    Article  PubMed  CAS  Google Scholar 

  71. Brown JN, Wallet MA, Drastins B, Sarracino D, Goodenow MM (2009) Proteome bioprofiles distinguish between M1 priming and activation states in human macrophages. J Leukoc Biol 87:655–662

    Article  Google Scholar 

  72. Melillo AA, Mahawar M, Sellati JJ, Malik M, Metzger DW, Melendez JA, Bakshi CS (2009) Identification of Francisella tularensis live vaccine strain CuZn superoxide dismutase as critical for resistance to extracellularly generated reactive oxygen species. J Bacteriol 191:6447–6456

    Article  PubMed  CAS  Google Scholar 

  73. Wehrly TD, Chong A, Virtaneva K, Sturdevant DE, Chile R, Edwards JA, Brouwer D, Nair V, Fischer ER, Wicke L, Curda AJ, Kuppko JJ III, Martens C, Crane DD, Bosio CM, Porcella SF, Celli J (2009) Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside microphages. Cell Microbiol 11:1128–1150

    Article  PubMed  CAS  Google Scholar 

  74. Lindgren H, Stenmark S, Chen W, Tarnvik A, Sjostedt A (2004) Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis. Infect Immun 72:71172–77182

    Article  Google Scholar 

  75. Al-Khodor A, Kwalk YA (2010) Triggering Ras signaling by intracellular Francisella tularensis through recruitment of PKCα and β1 to the SOS2/GrB2 complex is essential for bacterial proliferation in the cytosol. Cell Microbiol 12:1604–16021. doi:10.1111/j.1462-5822.2010.01494.x

    Article  PubMed  CAS  Google Scholar 

  76. Twine SM, Mykytczuk NCS, Petit MD, Shen H, Sjostedt A, Conlan JW, Kelly JF (2006) In vivo proteomic analysis of the intracellular bacterial pathogen, Francisella tularensis, isolated form mouse spleen. Biochem Biophys Res Commun 345:1621–1633

    Article  PubMed  CAS  Google Scholar 

  77. Smejkal GB, Robinson MH, Lawrence NP, Tao F, Saravis CA, Schumacher RT (2006) Increased protein yields from Escherichia coli using pressure-cycling technology. J Biomol Tech 17:173–175

    PubMed  Google Scholar 

  78. Yang F, Wu S, Senoien DL, Zhao R, Monroe ME, Gritsenko MA, Purvine SO, Polpitiya AD, Tolic N, Zhang Q, Noreck AD, Orton DJ, Moore RJ, Tang K, Anderson GA, Pasa-Tolic L, Camp DG II, Smith RD (2009) Combine pulsed-Q dissociation and electron transfer dissociation for identification and quantification of iTRAQ-labeled phosphopeptides. Anal Chem 81:4137–4143

    Article  PubMed  CAS  Google Scholar 

  79. Tenga MJ, Lazar IM (2011) Impact of peptide modifications on the isobaric tags for relative and absolute quantitation method accuracy. Anal Chem 83:701–707

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

The inclusion of specific product(s), product names, or corporate names in this article are for illustrative purposes only and does not imply nor constitute preference nor endorsement of a product or corporation by the author, USAMRIID, Department of the Army, or United States government. The views purported in this article reflect those of the author and do not reflect the views of USAMRIID, the U.S. Army, or U.S. government.

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Authors and Affiliations

  1. Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA

    Harry B. Hines

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  1. Harry B. Hines
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Correspondence to Harry B. Hines .

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  1. Physics & Life Sciences Directorate, Biosciences & Biotechnology Div., Lawrence Livermore National Laboratory, East Avenue 7000, Livermore, 94551, California, USA

    Ali Navid

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Hines, H.B. (2012). Microbial Proteomics Using Mass Spectrometry. In: Navid, A. (eds) Microbial Systems Biology. Methods in Molecular Biology, vol 881. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-827-6_7

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  • DOI: https://doi.org/10.1007/978-1-61779-827-6_7

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  • Publisher Name: Humana Press, Totowa, NJ

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