Skip to main content
SpringerLink
Log in
Book cover

Microbial Proteomics pp 231–247Cite as

Phosphopeptide Enrichment from Bacterial Samples Utilizing Titanium Oxide Affinity Chromatography

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1841))

Abstract

Mass spectrometry (MS)-based proteomics detected hundreds of phosphorylation sites on serine, threonine and tyrosine in numerous bacterial proteins, firmly establishing the presence and importance of this posttranslational modification in prokaryotes. Recent biological follow up of these results revealed that vital processes in bacterial cell, such as cell division, differentiation, spore germination and persistence, are regulated by protein phosphorylation, raising the need to study this modification on a global scale under additional physiological conditions. Due to low abundance and low stoichiometric levels of protein phosphorylation, initial protocols for phosphopeptide enrichment and analysis required relatively high amounts of starting material, extensive fractionation and MS measurement time. Here we present a protocol for phosphopeptide enrichment and detection based on TiO2 chromatography and high resolution MS that enables in-depth detection and quantification of phosphorylation sites from significantly lower amounts of starting material and in a fraction of MS measurement time.

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Mitrophanov AY, Groisman EA (2008) Signal integration in bacterial two-component regulatory systems. Genes Dev 22(19):2601–2611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Deutscher J, Saier MH Jr (2005) Ser/Thr/Tyr protein phosphorylation in bacteria - for long time neglected, now well established. J Mol Microbiol Biotechnol 9(3–4):125–131

    Article  CAS  PubMed  Google Scholar 

  3. Dworkin J (2015) Ser/Thr phosphorylation as a regulatory mechanism in bacteria. Curr Opin Microbiol 24(0):47–52

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wehenkel A, Bellinzoni M, Grana M, Duran R, Villarino A, Fernandez P, Andre-Leroux G, England P, Takiff H, Cervenansky C, Cole ST, Alzari PM (2008) Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential. Biochim Biophys Acta 1784(1):193–202

    Article  CAS  PubMed  Google Scholar 

  5. Kannan N, Taylor SS, Zhai Y, Venter JC, Manning G (2007) Structural and functional diversity of the microbial kinome. PLoS Biol 5(3):e17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Petranovic D, Michelsen O, Zahradka K, Silva C, Petranovic M, Jensen PR, Mijakovic I (2007) Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication. Mol Microbiol 63(6):1797–1805

    Article  CAS  PubMed  Google Scholar 

  7. Shah IM, Laaberki MH, Popham DL, Dworkin J (2008) A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments. Cell 135(3):486–496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Klein G, Dartigalongue C, Raina S (2003) Phosphorylation-mediated regulation of heat shock response in Escherichia coli. Mol Microbiol 48(1):269–285

    Article  CAS  PubMed  Google Scholar 

  9. Lacour S, Bechet E, Cozzone AJ, Mijakovic I, Grangeasse C (2008) Tyrosine phosphorylation of the UDP-glucose dehydrogenase of Escherichia coli is at the crossroads of colanic acid synthesis and polymyxin resistance. PLoS One 3(8):e3053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Molle V, Kremer L (2010) Division and cell envelope regulation by Ser/Thr phosphorylation: mycobacterium shows the way. Mol Microbiol 75(5):1064–1077

    Article  CAS  PubMed  Google Scholar 

  11. Fleurie A, Lesterlin C, Manuse S, Zhao C, Cluzel C, Lavergne JP, Franz-Wachtel M, Macek B, Combet C, Kuru E, VanNieuwenhze MS, Brun YV, Sherratt D, Grangeasse C (2014) MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae. Nature 516(7530):259–262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fleurie A, Manuse S, Zhao C, Campo N, Cluzel C, Lavergne JP, Freton C, Combet C, Guiral S, Soufi B, Macek B, Kuru E, VanNieuwenhze MS, Brun YV, Di Guilmi AM, Claverys JP, Galinier A, Grangeasse C (2014) Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division. PLoS Genet 10(4):e1004275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Morona JK, Miller DC, Morona R, Paton JC (2004) The effect that mutations in the conserved capsular polysaccharide biosynthesis genes cpsA, cpsB, and cpsD have on virulence of Streptococcus pneumoniae. J Infect Dis 189(10):1905–1913

    Article  CAS  PubMed  Google Scholar 

  14. Basell K, Otto A, Junker S, Zuhlke D, Rappen GM, Schmidt S, Hentschker C, Macek B, Ohlsen K, Hecker M, Becher D (2014) The phosphoproteome and its physiological dynamics in Staphylococcus aureus. Int J Med Microbiol 304(2):121–132

    Article  CAS  PubMed  Google Scholar 

  15. Schumacher MA, Piro KM, Xu W, Hansen S, Lewis K, Brennan RG (2009) Molecular mechanisms of HipA-mediated multidrug tolerance and its neutralization by HipB. Science 323(5912):396–401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Germain E, Castro-Roa D, Zenkin N, Gerdes K (2013) Molecular mechanism of bacterial persistence by HipA. Mol Cell 52(2):248–254

    Article  CAS  PubMed  Google Scholar 

  17. Lapek JD, Tombline G, Friedman AE (2010) Mass spectrometry detection of histidine phosphorylation on NM23-H1. J Proteome Res 10(2):751–755

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zu XL, Besant PG, Imhof A, Attwood PV (2007) Mass spectrometric analysis of protein histidine phosphorylation. Amino Acids 32(3):347–357

    Article  CAS  PubMed  Google Scholar 

  19. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362

    Article  CAS  PubMed  Google Scholar 

  20. Manza LL, Stamer SL, Ham AJ, Codreanu SG, Liebler DC (2005) Sample preparation and digestion for proteomic analyses using spin filters. Proteomics 5(7):1742–1745

    Article  CAS  PubMed  Google Scholar 

  21. Soufi B, Macek B (2015) Global analysis of bacterial membrane proteins and their modifications. Int J Med Microbiol 305(2):203–208

    Article  CAS  PubMed  Google Scholar 

  22. Soufi B, Macek B (2014) Stable isotope labeling by amino acids applied to bacterial cell culture. Methods Mol Biol 1188:9–22

    Article  CAS  PubMed  Google Scholar 

  23. Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221

    Article  CAS  PubMed  Google Scholar 

  24. Posewitz MC, Tempst P (1999) Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal Chem 71(14):2883–2892

    Article  CAS  PubMed  Google Scholar 

  25. Pinkse MWH, Uitto PM, Hilhorst MJ, Ooms B, Heck AJR (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-nanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76(14):3935–3943

    Article  CAS  PubMed  Google Scholar 

  26. Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJD (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4(7):873–886

    Article  CAS  PubMed  Google Scholar 

  27. Blagoev B, Ong SE, Kratchmarova I, Mann M (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat Biotechnol 22(9):1139–1145

    Article  CAS  PubMed  Google Scholar 

  28. Schmelzle K, Kane S, Gridley S, Lienhard GE, White FM (2006) Temporal dynamics of tyrosine phosphorylation in insulin signaling. Diabetes 55(8):2171–2179

    Article  CAS  PubMed  Google Scholar 

  29. Hansen AM, Chaerkady R, Sharma J, Diaz-Mejia JJ, Tyagi N, Renuse S, Jacob HK, Pinto SM, Sahasrabuddhe NA, Kim MS, Delanghe B, Srinivasan N, Emili A, Kaper JB, Pandey A (2013) The Escherichia coli phosphotyrosine proteome relates to core pathways and virulence. PLoS Pathog 9(6):e1003403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, Li JX, Cohn MA, Cantley LC, Gygi SP (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101(33):12130–12135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kelstrup CD, Jersie-Christensen RR, Batth TS, Arrey TN, Kuehn A, Kellmann M, Olsen JV (2014) Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer. J Proteome Res 13(12):6187–6195

    Article  CAS  PubMed  Google Scholar 

  32. Ishihama Y, Rappsilber J, Andersen JS, Mann M (2002) Microcolumns with self-assembled particle frits for proteomics. J Chromatogr A 979(1–2):233–239

    Article  CAS  PubMed  Google Scholar 

  33. Hillenkamp F, Karas M, Beavis RC, Chait BT (1991) Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal Chem 63(24):1193A–1203A

    Article  CAS  PubMed  Google Scholar 

  34. Ahmed FE (2008) Utility of mass spectrometry for proteome analysis: part I. Conceptual and experimental approaches. Expert Rev Proteomics 5(6):841–864

    Article  CAS  PubMed  Google Scholar 

  35. Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4(12):2010–2021

    Article  CAS  PubMed  Google Scholar 

  36. Schroeder MJ, Shabanowitz J, Schwartz JC, Hunt DF, Coon JJ (2004) A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal Chem 76(13):3590–3598

    Article  CAS  PubMed  Google Scholar 

  37. Olsen JV, Macek B, Lange O, Makarov A, Horning S, Mann M (2007) Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods 4(9):709–712

    Article  CAS  PubMed  Google Scholar 

  38. Chi A, Huttenhower C, Geer LY, Coon JJ, Syka JE, Bai DL, Shabanowitz J, Burke DJ, Troyanskaya OG, Hunt DF (2007) Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc Natl Acad Sci U S A 104(7):2193–2198

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Good DM, Wirtala M, McAlister GC, Coon JJ (2007) Performance characteristics of electron transfer dissociation mass spectrometry. Mol Cell Proteomics 6(11):1942–1951

    Article  CAS  PubMed  Google Scholar 

  40. Frese CK, Altelaar AF, Hennrich ML, Nolting D, Zeller M, Griep-Raming J, Heck AJ, Mohammed S (2011) Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos. J Proteome Res 10(5):2377–2388

    Article  CAS  PubMed  Google Scholar 

  41. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372

    Article  CAS  PubMed  Google Scholar 

  42. The Universal Protein Resource (UniProt) (2009) Nucleic Acids Res 37(Database issue):D169–D174

    Google Scholar 

  43. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18):3551–3567

    Article  CAS  Google Scholar 

  44. Yates JR 3rd, Eng JK, McCormack AL, Schieltz D (1995) Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem 67(8):1426–1436

    Article  CAS  PubMed  Google Scholar 

  45. Geer LY, Markey SP, Kowalak JA, Wagner L, Xu M, Maynard DM, Yang X, Shi W, Bryant SH (2004) Open mass spectrometry search algorithm. J Proteome Res 3(5):958–964

    Article  CAS  PubMed  Google Scholar 

  46. Elias JE, Gygi SP (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4(3):207–214

    Article  CAS  PubMed  Google Scholar 

  47. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127(3):635–648

    Article  CAS  PubMed  Google Scholar 

  48. Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24(10):1285–1292

    Article  CAS  PubMed  Google Scholar 

  49. Ravikumar V, Macek B, Mijakovic I (2016) Resources for assignment of phosphorylation sites on peptides and proteins. Methods Mol Biol 1355:293–306

    Article  CAS  PubMed  Google Scholar 

  50. Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, Mann M (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4(5):698–705

    Article  CAS  PubMed  Google Scholar 

  51. Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10(4):1794–1805

    Article  CAS  PubMed  Google Scholar 

  52. Tyanova S, Mann M, Cox J (2014) MaxQuant for in-depth analysis of large SILAC datasets. In: Warscheid B (ed) Stable isotope labeling by amino acids in cell culture (SILAC), Methods in molecular biology, vol 1188. Springer, New York, pp 351–364

    Google Scholar 

Download references

Acknowledgments

We thank Dr. Olaf Voolstra for critical reading of the manuscript. Our work is supported by the SFB766 of the Deutsche Forschungsgemeinschaft and PRIME-XS consortium.

Author information

Authors and Affiliations

  1. Proteome Center Tübingen, University of Tübingen, Tübingen, Germany

    Boumediene Soufi, Christoph Täumer, Maja Semanjski & Boris Macek

Authors
  1. Boumediene Soufi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christoph Täumer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maja Semanjski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Boris Macek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Boris Macek .

Editor information

Editors and Affiliations

  1. Department of Microbial Proteomics, Institute for Microbiology, University Greifswald, Greifswald, Germany

    Dörte Becher

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Soufi, B., Täumer, C., Semanjski, M., Macek, B. (2018). Phosphopeptide Enrichment from Bacterial Samples Utilizing Titanium Oxide Affinity Chromatography. In: Becher, D. (eds) Microbial Proteomics. Methods in Molecular Biology, vol 1841. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8695-8_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8695-8_16

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8693-4

  • Online ISBN: 978-1-4939-8695-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation