Skip to main content

Analysis of Methylation, Acetylation, and Other Modifications in Bacterial Ribosomal Proteins

  • Protocol
  • First Online:
Post-Translational Modification of Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1934))

Abstract

A wide variety of posttranslational modifications of expressed proteins are known to occur in living organisms (Krishna R, Wold F. Post-translational modification of proteins. In: Meister A (ed) Advances in enzymology and related areas of molecular biology. Wiley, New York, 1993, pp 265–296). Although their presence in an organism cannot be predicted from the genome, these modifications can play critical roles in protein structure and function. The identification of posttranslational modifications is critical to our understanding of the functions of proteins involved in important biological pathways and mass spectrometry offers a fast, accurate method for observing them. A combined top-down/bottom-up approach can be used for identification and localization of posttranslational modifications of ribosomal proteins. This chapter describes procedures for analyzing Escherichia coli ribosomal proteins and their modifications by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. It also covers the analysis of gram-negative Caulobacter crescentus and gram-positive Bacillus subtilis ribosomal proteins by electrospray quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Confirmation of the occurrence and localization of PTMs is obtained through mass spectrometric analysis of tryptic peptides.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Krishna R, Wold F (1993) Post-translational modification of proteins. In: Meister A (ed) Advances in enzymology and related areas of molecular biology. Wiley, New York, pp 265–296

    Google Scholar 

  2. Running WE, Ravipaty S, Karty J, Reilly JP (2007) A top-down/bottom-up study of the ribosomal proteins of Caulobacter crescentus. J Proteome Res 6:337–347

    Article  CAS  Google Scholar 

  3. Lauber MA, Rappsilber J, Reilly JP (2012) Observing the binding site and dynamics of protein S1 on a bacterial ribosome with cross-linking and mass spectrometry. Mol Cell Proteomics 11:1965–1976

    Article  Google Scholar 

  4. Spedding G (1990) Isolation of ribosomes from prokaryotes, eukaryotes, and organelles. In: Rickwood D, Hames BD (eds) Ribosomes and protein synthesis, a practical approach. Oxford University Press, New York, pp 4–7

    Google Scholar 

  5. Ramakrishnan V, White S (1998) Ribosomal protein structures: insights into the architecture, machinery and evolution of the ribosome. Trends Biochem Sci 23:208–212

    Article  CAS  Google Scholar 

  6. Podzorski RP, Persing DH (1995) Molecular detection and identification of microorganisms. In: Murray RD, et al. Manual of clinical microbiology. ASM Press, Washington, DC, pp 130–157

    Google Scholar 

  7. Woese CR (1987) Bacterial evolution. Microbiol Rev 51:221–271

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Noller HF, Nomura M (1996) Ribosomes. In: Neidhardt FD (ed) Escherichia coli and Salmonella, vol 1. ASM Press, pp 167–182, Washington, DC

    Google Scholar 

  9. Wittmann HG (1982) Components of bacterial ribosomes. Annu Rev Biochem 51:155–183

    Article  CAS  Google Scholar 

  10. Van Buskirk J, Kirsch W (1978) gamma-Carboxyglutamic acid in eukaryotic and prokaryotic ribosomes. Biochem Biophys Res Commun 82:1329–1331

    Article  Google Scholar 

  11. Kowalak J, Walsh K (1996) Beta-methylthio-aspartic acid: identification of a novel posttranslational modification in ribosomal protein S12 from Escherichia coli. Protein Sci 5:1625–1632

    Article  CAS  Google Scholar 

  12. Lauber MA, Running WE, Reilly JP (2009) B. subtilis ribosomal proteins: structural homology and post-translational modifications. J. Proteome Res 8:4193–4206

    Article  CAS  Google Scholar 

  13. Running WE, Reilly JP (2009) Ribosomal proteins of Deinococcus radiodurans: their solvent accessibility and reactivity. J Proteome Res 8:1228–1246

    Article  CAS  Google Scholar 

  14. Neidhardt FC, Ingraham JL, Schaechter M (1990) Physiology of the bacterial cell. Sinauer Associates, MA

    Google Scholar 

  15. Geyl D, Bock A, Isono K (1981) An improved method for two-dimensional gel-electrophoresis: analysis of mutationally altered ribosomal proteins of Escherichia coli. Mol Gen Genet 181:309–312

    Article  CAS  Google Scholar 

  16. Datta DB, Changchien L, Nierras CR et al (1988) Identification of Escherichia coli ribosomal proteins by an alternative two-dimensional electrophoresis system. Anal Biochem 173:241–245

    Article  CAS  Google Scholar 

  17. Fenn JB, Mann M, Meng CK et al (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246:64–71

    Article  CAS  Google Scholar 

  18. Karas M, Bachmann D, Bahr U, Hillenkamp F (1987) Matrix-assisted ultraviolet laser desorption of non-volatile compounds. Int J Mass Spectrom Ion Proc 78:53–68

    Article  CAS  Google Scholar 

  19. Colby S, King T, Reilly J (1994) Improving the resolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by exploiting the correlation between ion position and velocity. Rapid Commun Mass Spectrom 8:865–868

    Article  CAS  Google Scholar 

  20. Whittal RM, Li L (1995) High-resolution matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer. Anal Chem 67:1950–1954

    Article  CAS  Google Scholar 

  21. Brown RS, Lennon JJ (1995) Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometer. Anal Chem 67:1998–2003

    Article  CAS  Google Scholar 

  22. Bremer H, Dennis PP (1996) Modulation of chemical composition and other parameters of the cell by growth rate. In: Neidhardt FD (ed) Escherichia coli and Salmonella, vol 2. ASM Press, Washington, DC, pp 1553–1569

    Google Scholar 

  23. Arnold R, Reilly J (1999) Observation of Escherichia coli ribosomal proteins and their posttranslational modifications by mass spectrometry. Anal Biochem 269:105–112

    Article  CAS  Google Scholar 

  24. Arnold R, Polevoda B, Reilly J, Sherman F (1999) The action of N-terminal acetyltransferases on yeast ribosomal proteins. J Biol Chem 274:37035–37040

    Article  CAS  Google Scholar 

  25. Karty JA, Running WE, Reilly JP (2007) Two dimensional liquid phase separations of proteins using online fractionation and concentration between chromatographic dimensions. J Chromatogr B Analyt Technol Biomed Life Sci 847:103–113

    Article  CAS  Google Scholar 

  26. McEwen CN, Larsen BS (1997) Electrospray ionization on quadrupole and magnetic-sector mass spectrometers. In: Cole RB (ed) Electrospray ionization mass spectrometry. Wiley, pp 171–202

    Google Scholar 

  27. Roth MJ, Forbes AJ, Boyne MT II et al (2005) Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry. Mol Cell Proteomics 4:1002–1008

    Article  CAS  Google Scholar 

  28. Jebanathirajah JA, Pittman JL, Thomson BA et al (2005) Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins. J Am Soc Mass Spectrom 16:1985–1999

    Article  CAS  Google Scholar 

  29. Macek B, Waanders LF, Olsen JV, Mann M (2006) Top-down protein sequencing and MS3 on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer. Mol Cell Proteomics 5:949–958

    Article  CAS  Google Scholar 

  30. Ge Y, Lawhorn BG, ElNaggar M et al (2002) Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry. J Am Chem Soc 124:672–678

    Article  CAS  Google Scholar 

  31. Zubarev RA (2004) Electron-capture dissociation tandem mass spectrometry. Curr Opin Biotechnol 15:12–16

    Article  CAS  Google Scholar 

  32. Cooper HJ, Hakansson K, Marshall AG (2005) The role of electron capture dissociation in biomolecular analysis. Mass Spectrom Rev 24:201–222

    Article  CAS  Google Scholar 

  33. Reilly JP (2009) Ultraviolet photofragmentation of biomolecular ions. Mass Spectrom Rev 28:425–447

    Article  CAS  Google Scholar 

  34. Shaw JB, Li W, Holden DD et al (2013) Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation. J Am Chem Soc 135:12646–12651

    Article  CAS  Google Scholar 

  35. Meng F, Du Y, Miller LM et al (2004) Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics. Anal Chem 76:2852–2858

    Article  CAS  Google Scholar 

  36. Patrie SM, Ferguson JT, Robinson DE et al (2006) Top down mass spectrometry of <60-kDa proteins from Methanosarcina acetivorans using quadrupole FRMS with automated octopole collisionally activated dissociation. Mol Cell Proteomics 5:14–25

    Article  CAS  Google Scholar 

  37. Whitelegge JP, Laganowsky A, Nishio J et al (2006) Sequencing covalent modifications of membrane proteins. J Exp Bot 57:1515–1522

    Article  CAS  Google Scholar 

  38. Tran JC, Zamborg L, Ahlf DR et al (2011) Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature 480:254–258

    Article  CAS  Google Scholar 

  39. Chen SH, Russell WK, Russell DH (2013) Combining chemical labeling, bottom-up and top-down ion-mobility mass spectrometry to identify metal-binding sites of partially metalated metallothionein. Anal Chem 85:3229–3237

    Article  CAS  Google Scholar 

  40. Bergmann U, Ahrends R, Neumann B et al (2012) Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging. Anal Chem 84:5268–5275

    Article  CAS  Google Scholar 

  41. Bouchal P, Dvorakova M, Scherl A et al (2013) Intact protein profiling in breast cancer biomarker discovery: protein identification issue and the solutions based on 3D protein separation, bottom-up and top-down mass spectrometry. Proteomics 13:1053–1058

    Article  CAS  Google Scholar 

  42. Ahlf DR, Thomas PM, Kelleher NL (2013) Developing top down proteomics to maximize proteome and sequence coverage from cells and tissues. Curr Opin Chem Biol 17:787–794

    Article  CAS  Google Scholar 

  43. Catherman AD, Durbin KR, Ahlf DR et al (2013) Large-scale top-down proteomics of the human proteome: membrane proteins, mitochondria, and senescence. Mol Cell Proteomics 12:3465–3473

    Article  CAS  Google Scholar 

  44. Drabik A, Bodzon-Kulakowska A, Suder P (2012) Application of the ETD/PTR reactions in top-down proteomics as a faster alternative to bottom-up nano-LC-MS/MS protein identification. J Mass Spectrom 47:1347–1352

    Article  CAS  Google Scholar 

  45. Warren ME, Brockman AH, Orlando R (1998) On-probe solid-phase extraction/MALDI-MS using ion-pairing interactions for the cleanup of peptides and proteins. Anal Chem 70:3757–3761

    Article  CAS  Google Scholar 

  46. Sherman F, Stewart JW, Tsunasawa S (1985) Methionine or not methionine at the beginning of a protein. BioEssays 3:27–31

    Article  CAS  Google Scholar 

  47. Diedrich G, Burkhardt N, Nierhaus KH (1997) Large-scale isolation of proteins of the large subunit from Escherichia coli ribosomes. Protein Expr Purif 10:42–50

    Article  CAS  Google Scholar 

  48. Champney WS (1990) Reversed-phase chromatography of Escherichia coli ribosomal proteins. Correlation of retention time with chain length and hydrophobicity. J Chromatogr 522:163–170

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work has been supported by National Science Foundation grant CHE-1012855 to J.P.R. and National Institutes of Health grant U54 GM105816 to the Protein Translation Research Network.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to James P. Reilly .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Arnold, R.J., Saraswat, S., Reilly, J.P. (2019). Analysis of Methylation, Acetylation, and Other Modifications in Bacterial Ribosomal Proteins. In: Kannicht, C. (eds) Post-Translational Modification of Proteins. Methods in Molecular Biology, vol 1934. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9055-9_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9055-9_18

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9053-5

  • Online ISBN: 978-1-4939-9055-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics