Skip to main content
SpringerLink
Log in

Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Owing to its broad biological significance, the large-scale analysis of protein phosphorylation is more and more getting into the focus of proteomic research. Thousands of phosphopeptides can nowadays be identified using state-of-the-art tandem mass spectrometers in conjunction with sequence database searching, but localizing the phosphate group to a particular amino acid in the peptide sequence is often still difficult. Using 180 individually synthesized phosphopeptides with precisely known phosphorylation sites (p-sites), we have assessed the merits of the Mascot Delta Score (MD score) for the assignment of phosphorylation sites from tandem mass spectra (MS/MS) generated on four different matrix-assisted laser desorption ionization (MALDI) mass spectrometers including tandem time-of-flight (TOF/TOF), quadrupole time-of-flight, and ion trap mass analyzers. The results show that phosphorylation site identification is generally possible with false localization rates of about 10%. However, a comparison to previous work also revealed that phosphorylation site determination by MALDI MS/MS is less accurate than by ESI-MS/MS particularly if several and/or adjacent possible phosphorylation acceptor sites exist in a peptide sequence. We are making the tandem MS spectra and phosphopeptide collection available to the community so that scientists may adapt the MD scores reported here to their analytical environment and so that informatics developers may integrate the MD score into proteomic data analysis pipelines.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

CID:

Collision-induced dissociation

ESI:

Electrospray ionization

ETD:

Electron transfer dissociation

ETDSA:

Electron transfer dissociation with supplemental activation

FLR:

False localization rate

HCD:

Higher energy collision-induced dissociation

LC-MS/MS:

Liquid chromatography–tandem mass spectrometry

MALDI:

Matrix-assisted laser desorption ionization

MD score:

Mascot Delta score

Mgf:

Mascot generic format

MSA:

Multistage activation

PKL:

Peak list file

PSD:

Post-source decay

p-sites:

Phosphorylation sites

PTMs:

Posttranslational modifications

QTOF:

Quadrupole time of flight

TOF/TOF:

Tandem time of flight

References

  1. Mallick P, Kuster B (2010) Proteomics: a pragmatic perspective. Nat Biotechnol 28(7):695–709. doi:10.1038/nbt.1658

    Article  CAS  Google Scholar 

  2. Pinkse MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ (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. doi:10.1021/ac0498617

    Article  CAS  Google Scholar 

  3. Thingholm TE, Jensen ON, Robinson PJ, Larsen MR (2008) SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol Cell Proteomics 7(4):661–671. doi:10.1074/mcp.M700362-MCP200

    CAS  Google Scholar 

  4. Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, Li J, 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. doi:10.1073/pnas.0404720101

    Article  CAS  Google Scholar 

  5. 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. doi:10.1016/j.cell.2006.09.026

    Article  CAS  Google Scholar 

  6. Swaney DL, Wenger CD, Thomson JA, Coon JJ (2009) Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A 106(4):995–1000. doi:10.1073/pnas.0811964106

    Article  CAS  Google Scholar 

  7. Zhang Y, Wolf-Yadlin A, Ross PL, Pappin DJ, Rush J, Lauffenburger DA, White FM (2005) Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol Cell Proteomics 4(9):1240–1250. doi:10.1074/mcp.M500089-MCP200

    Article  CAS  Google Scholar 

  8. Nichols AM, White FM (2009) Manual validation of peptide sequence and sites of tyrosine phosphorylation from MS/MS spectra. Methods Mol Biol 492:143–160. doi:10.1007/978-1-59745-493-3_8

    Article  CAS  Google Scholar 

  9. Bailey CM, Sweet SM, Cunningham DL, Zeller M, Heath JK, Cooper HJ (2009) SLoMo: automated site localization of modifications from ETD/ECD mass spectra. J Proteome Res 8(4):1965–1971. doi:10.1021/pr800917p

    Article  CAS  Google Scholar 

  10. 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. doi:10.1038/nbt1240

    Article  CAS  Google Scholar 

  11. Payne SH, Yau M, Smolka MB, Tanner S, Zhou H, Bafna V (2008) Phosphorylation-specific MS/MS scoring for rapid and accurate phosphoproteome analysis. J Proteome Res 7(8):3373–3381. doi:10.1021/pr800129m

    Article  CAS  Google Scholar 

  12. Ruttenberg BE, Pisitkun T, Knepper MA, Hoffert JD (2008) PhosphoScore: an open-source phosphorylation site assignment tool for MSn data. J Proteome Res 7(7):3054–3059. doi:10.1021/pr800169k

    Article  CAS  Google Scholar 

  13. Schlosser A, Vanselow JT, Kramer A (2007) Comprehensive phosphorylation site analysis of individual phosphoproteins applying scoring schemes for MS/MS data. Anal Chem 79(19):7439–7449. doi:10.1021/ac0707784

    Article  CAS  Google Scholar 

  14. Wan Y, Cripps D, Thomas S, Campbell P, Ambulos N, Chen T, Yang A (2008) PhosphoScan: a probability-based method for phosphorylation site prediction using MS2/MS3 pair information. J Proteome Res 7(7):2803–2811. doi:10.1021/pr700773p

    Article  CAS  Google Scholar 

  15. Baker PR, Trinidad JC, Chalkley RJ (2011) Modification site localization scoring integrated into a search engine. Mol Cell Proteomics 10:M111.008078. doi:10.1074/mcp.M111.008078

    Article  Google Scholar 

  16. Lu B, Ruse C, Xu T, Park SK, Yates J 3rd (2007) Automatic validation of phosphopeptide identifications from tandem mass spectra. Anal Chem 79(4):1301–1310. doi:10.1021/ac061334v

    Article  CAS  Google Scholar 

  17. 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. doi:10.1002/(SICI)1522-2683(19991201)20:18<3551

    Article  CAS  Google Scholar 

  18. Savitski MM, Lemeer S, Boesche M, Lang M, Mathieson T, Bantscheff M, Kuster B (2011) Confident phosphorylation site localization using the Mascot Delta Score. Mol Cell Proteomics 10(2):M110 003830. doi:10.1074/mcp.M110.003830

  19. 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  Google Scholar 

  20. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246(4926):64–71

    Article  CAS  Google Scholar 

  21. Karas M, Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60(20):2299–2301

    Article  CAS  Google Scholar 

  22. Bennett KL, Stensballe A, Podtelejnikov AV, Moniatte M, Jensen ON (2002) Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry. J Mass Spectrom 37(2):179–190. doi:10.1002/jms.271

    Article  CAS  Google Scholar 

  23. Condina MR, Gustafsson JO, Klingler-Hoffmann M, Bagley CJ, McColl SR, Hoffmann P (2010) EZYprep LC-coupled MALDI-TOF/TOF MS: an improved matrix spray application for phosphopeptide characterisation. Proteomics 10(13):2516–2530. doi:10.1002/pmic.200900800

    Article  CAS  Google Scholar 

  24. Schmidt A, Csaszar E, Ammerer G, Mechtler K (2008) Enhanced detection and identification of multiply phosphorylated peptides using TiO2 enrichment in combination with MALDI TOF/TOF MS. Proteomics 8(21):4577–4592. doi:10.1002/pmic.200800279

    Article  CAS  Google Scholar 

  25. Griffin PR, MacCoss MJ, Eng JK, Blevins RA, Aaronson JS, Yates JR 3rd (1995) Direct database searching with MALDI-PSD spectra of peptides. Rapid Commun Mass Spectrom 9(15):1546–1551. doi:10.1002/rcm.1290091515

    Article  CAS  Google Scholar 

  26. Lehmann WD, Kruger R, Salek M, Hung CW, Wolschin F, Weckwerth W (2007) Neutral loss-based phosphopeptide recognition: a collection of caveats. J Proteome Res 6(7):2866–2873. doi:10.1021/pr060573w

    Article  CAS  Google Scholar 

  27. Boersema PJ, Mohammed S, Heck AJ (2009) Phosphopeptide fragmentation and analysis by mass spectrometry. J Mass Spectrom 44(6):861–878. doi:10.1002/jms.1599

    Article  CAS  Google Scholar 

  28. Mischerikow N, Altelaar AF, Navarro JD, Mohammed S, Heck A (2010) Comparative assessment of site assignments in CID and ETD spectra of phosphopeptides discloses limited relocation of phosphate groups. Mol Cell Proteomics 9:2140–2148. doi:10.1074/mcp.M900619-MCP200

    Article  CAS  Google Scholar 

  29. Bantscheff M, Eberhard D, Abraham Y, Bastuck S, Boesche M, Hobson S, Mathieson T, Perrin J, Raida M, Rau C, Reader V, Sweetman G, Bauer A, Bouwmeester T, Hopf C, Kruse U, Neubauer G, Ramsden N, Rick J, Kuster B, Drewes G (2007) Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat Biotechnol 25(9):1035–1044. doi:10.1038/nbt1328

    Article  CAS  Google Scholar 

  30. Gnad F, Ren S, Cox J, Olsen JV, Macek B, Oroshi M, Mann M (2007) PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol 8(11):R250. doi:10.1186/gb-2007-8-11-r250

    Article  Google Scholar 

  31. Suckau D, Resemann A, Schuerenberg M, Hufnagel P, Franzen J, Holle A (2003) A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal Bioanal Chem 376(7):952–965. doi:10.1007/s00216-003-2057-0

    Article  CAS  Google Scholar 

  32. 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. doi:10.1038/nmeth1019

    Article  CAS  Google Scholar 

  33. Medzihradszky KF, Campbell JM, Baldwin MA, Falick AM, Juhasz P, Vestal ML, Burlingame AL (2000) The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer. Anal Chem 72(3):552–558

    Article  CAS  Google Scholar 

  34. Harvey DJ, Bateman RH, Bordoli RS, Tyldesley R (2000) Ionisation and fragmentation of complex glycans with a quadrupole time-of-flight mass spectrometer fitted with a matrix-assisted laser desorption/ionisation ion source. Rapid Commun Mass Spectrom 14(22):2135–2142. doi:10.1002/1097-0231(20001130)14:22<2135::AID-RCM143>3.0.CO;2-#

    Article  CAS  Google Scholar 

  35. Verhaert PD, Pinkse MW, Strupat K, Conaway MC (2010) Imaging of similar mass neuropeptides in neuronal tissue by enhanced resolution MALDI MS with an ion trap–Orbitrap hybrid instrument. Methods Mol Biol 656:433–449. doi:10.1007/978-1-60761-746-4_25

    Article  CAS  Google Scholar 

  36. Spengler B (2004) De novo sequencing, peptide composition analysis, and composition-based sequencing: a new strategy employing accurate mass determination by Fourier transform ion cyclotron resonance mass spectrometry. J Am Soc Mass Spectrom 15(5):703–714. doi:10.1016/j.jasms.2004.01.007

    Article  CAS  Google Scholar 

  37. Nagaraj N, D’Souza RC, Cox J, Olsen JV, Mann M (2010) Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation. J Proteome Res 9(12):6786–6794. doi:10.1021/pr100637q

    Article  CAS  Google Scholar 

  38. McAlister GC, Phanstiel DH, Brumbaugh J, Westphall MS, Coon JJ (2011) Higher-energy collision-activated dissociation without a dedicated collision cell. Mol Cell Proteomics 10(5):O111 009456. doi:10.1074/mcp.O111.009456

  39. Edelson-Averbukh M, Shevchenko A, Pipkorn R, Lehmann WD (2009) Gas-phase intramolecular phosphate shift in phosphotyrosine-containing peptide monoanions. Anal Chem 81(11):4369–4381. doi:10.1021/ac900244e

    Article  CAS  Google Scholar 

  40. Aguiar M, Haas W, Beausoleil SA, Rush J, Gygi SP (2010) Gas-phase rearrangements do not affect site localization reliability in phosphoproteomics data sets. J Proteome Res 9:3103–3107. doi:10.1021/pr1000225

    Article  CAS  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. doi:10.1038/nbt.1511

    Article  CAS  Google Scholar 

  42. Savitski MM, Mathieson T, Becher I, Bantscheff M (2010) H-Score, a mass accuracy driven rescoring approach for improved peptide identification in modification rich samples. J Proteome Res 9:5511–5516. doi:10.1021/pr1006813

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Andrea Hubauer for expert technical assistance.

Author information

Authors and Affiliations

  1. Chair of Proteomics and Bioanalytics, Technische Universität München, Emil Erlenmeyer Forum 5, 85354, Freising, Germany

    Simone Lemeer, Elena Kunold, Susan Klaeger & Bernhard Kuster

  2. Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany

    Monika Raabe & Henning Urlaub

  3. Waters Corporation, Atlas Park, Simmons Way, Manchester, M22 5PP, UK

    Mark W. Towers & Emmanuelle Claudes

  4. Thermo Fisher Scientific, Hanna-Kunath-Strasse 11, 28199, Bremen, Germany

    Tabiwang N. Arrey & Kerstin Strupat

  5. Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, Robert Koch Strasse 42, 37075, Göttingen, Germany

    Henning Urlaub

  6. Center for Integrated Protein Science Munich (CIPSM), Munich, Germany

    Bernhard Kuster

Authors
  1. Simone Lemeer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elena Kunold
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Susan Klaeger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Monika Raabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark W. Towers
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Emmanuelle Claudes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tabiwang N. Arrey
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kerstin Strupat
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Henning Urlaub
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Bernhard Kuster
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bernhard Kuster.

Additional information

Published in the 10th Anniversary Issue.

Simone Lemeer, Elena Kunold, and Susan Klaeger contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 3.14 mb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lemeer, S., Kunold, E., Klaeger, S. et al. Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score. Anal Bioanal Chem 402, 249–260 (2012). https://doi.org/10.1007/s00216-011-5469-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-011-5469-2

Keywords

Search

Navigation