Amino Acids

, Volume 40, Issue 2, pp 697–711 | Cite as

Observed peptide pI and retention time shifts as a result of post-translational modifications in multidimensional separations using narrow-range IPG-IEF

  • Johan Lengqvist
  • Hanna Eriksson
  • Marcus Gry
  • Kristina Uhlén
  • Christina Björklund
  • Bengt Bjellqvist
  • Per-Johan Jakobsson
  • Janne Lehtiö
Original Article


Modified peptides constitute a sub-population among the tryptic peptides analyzed in LC–MS based shotgun proteomics experiments. For larger proteomes including the human proteome, the tryptic peptide pool is very large, which necessitates some form of sample fractionation. By carefully choosing the sample fractionation and separation methods applied as shown here for the combination of narrow-range immobilized pH gradient isoelectric focusing (IPG-IEF) and nanoUPLC–MS, significantly increased information content can be achieved. Relatively low standard deviations were obtained for such multidimensional separations in terms of peptide pI (<0.05 pI units) and retention time (<0.3 min for a 350 min gradient) for a selection of highly complex proteomics samples. Using narrow-range IPG-IEF, experimental and predicted pI were in relative good agreement. However, based on our data, retention time prediction algorithms need further improvements in accuracy to match state-of-the-art reversed-phase chromatography performance. General trends of peptide pI shifts induced by common modifications including deamidations and N-terminal modifications are described. Deamidations of glutamine and asparagines shift peptide pI by approximately 1.5 pI units, making the peptides more acidic. Additionally, a novel pI shift (+~0.4 pI units) was found associated with dethiomethyl Met modifications. Further, the effects of these modifications as well as methionine oxidation were investigated in terms of experimentally observed retention time shifts in the chromatographic separation step. Clearly, post-translational modification-induced influences on peptide pI and retention time can be accurately and reproducibly measured using narrow-range IPG-IEF and high-performance nanoLC–MS. Even at modest mass accuracy (±50 ppm), the inclusion of peptide pI (±0.2 pI units) and/or retention time (±20 min) criteria are highly informative for human proteome analyses. The applications of using this information to identify post-translationally modified peptides and improve data analysis workflows are discussed.


Isoelectric focusing Post-translational modifications Retention time 



This study was supported by grants from the Department of Research and Development at the Karolinska University Hospital and Stockholm County Council, Sweden, the Cancer Society in Stockholm, Sweden, Swedish Cancer Society and the Swedish Research Counsel 2009-5083 and 2004-5259. Instrumentation was acquired through grants from Knut and Alice Wallenberg Foundation.

Supplementary material

726_2010_704_MOESM1_ESM.doc (58 kb)
Supplementary material 1 (DOC 58 kb)
726_2010_704_MOESM2_ESM.tif (129 kb)
Supplementary material 2 (TIFF 129 kb)


  1. Ahrne E, Muller M, Lisacek F (2009) Unrestricted identification of modified proteins using MS/MS. Proteomics 10(4):671–686CrossRefGoogle Scholar
  2. Anderson NL, Jackson A, Smith D, Hardie D, Borchers C, Pearson TW (2009) Siscapa peptide enrichment on magnetic beads using an in-line bead trap device. Mol Cell Proteomics 8(5):995–1005CrossRefPubMedGoogle Scholar
  3. Bellew M, Coram M, Fitzgibbon M, Igra M, Randolph T, Wang P, May D, Eng J, Fang R, Lin C, Chen J, Goodlett D, Whiteaker J, Paulovich A, McIntosh M (2006) A suite of algorithms for the comprehensive analysis of complex protein mixtures using high-resolution LC–MS. Bioinformatics 22(15):1902–1909CrossRefPubMedGoogle Scholar
  4. Berg M, Parbel A, Pettersen H, Fenyo D, Bjorkesten L (2006) Detection of artifacts and peptide modifications in liquid chromatography/mass spectrometry data using two-dimensional signal intensity map data visualization. Rapid Commun Mass Spectrom 20(10):1558–1562CrossRefPubMedGoogle Scholar
  5. Blondelle SE, Ostresh JM, Houghten RA, Perez-Paya E (1995) Induced conformational states of amphipathic peptides in aqueous/lipid environments. Biophys J 68(1):351–359CrossRefPubMedGoogle Scholar
  6. Browne CA, Bennett HP, Solomon S (1982) The isolation of peptides by high-performance liquid chromatography using predicted elution positions. Anal Biochem 124(1):201–208CrossRefPubMedGoogle Scholar
  7. Cargile BJ, Stephenson JL Jr (2004) An alternative to tandem mass spectrometry: isoelectric point and accurate mass for the identification of peptides. Anal Chem 76(2):267–275CrossRefPubMedGoogle Scholar
  8. Cargile BJ, Bundy JL, Freeman TW, Stephenson JL Jr (2004a) Gel based isoelectric focusing of peptides and the utility of isoelectric point in protein identification. J Proteome Res 3(1):112–119CrossRefPubMedGoogle Scholar
  9. Cargile BJ, Talley DL, Stephenson JL Jr (2004b) Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pi predictability of peptides. Electrophoresis 25(6):936–945CrossRefPubMedGoogle Scholar
  10. Cargile BJ, Sevinsky JR, Essader AS, Stephenson JL Jr, Bundy JL (2005) Immobilized pH gradient isoelectric focusing as a first-dimension separation in shotgun proteomics. J Biomol Tech 16(3):181–189PubMedGoogle Scholar
  11. Cargile BJ, Sevinsky JR, Essader AS, Eu JP, Stephenson JL Jr (2008) Calculation of the isoelectric point of tryptic peptides in the pH 3.5–4.5 range based on adjacent amino acid effects. Electrophoresis 29(13):2768–2778Google Scholar
  12. Craig R, Cortens JP, Beavis RC (2004) Open source system for analyzing, validating, and storing protein identification data. J Proteome Res 3(6):1234–1242CrossRefPubMedGoogle Scholar
  13. Desiere F, Deutsch EW, Nesvizhskii AI, Mallick P, King NL, Eng JK, Aderem A, Boyle R, Brunner E, Donohoe S, Fausto N, Hafen E, Hood L, Katze MG, Kennedy KA, Kregenow F, Lee H, Lin B, Martin D, Ranish JA, Rawlings DJ, Samelson LE, Shiio Y, Watts JD, Wollscheid B, Wright ME, Yan W, Yang L, Yi EC, Zhang H, Aebersold R (2005) Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry. Genome Biol 6(1):R9. doi: gb-2004-6-1-r9[pii]10.1186/gb-2004-6-1-r9
  14. Eriksson H, Lengqvist J, Hedlund J, Uhlen K, Orre LM, Bjellqvist B, Persson B, Lehtio J, Jakobsson PJ (2008) Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms. Proteomics 8(15):3008–3018CrossRefPubMedGoogle Scholar
  15. Essader AS, Cargile BJ, Bundy JL, Stephenson JL Jr (2005) A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics. Proteomics 5(1):24–34CrossRefPubMedGoogle Scholar
  16. Fenyö D, Beavis RC (2008) Informatics development: challenges and solutions for MALDI mass spectrometry. Mass Spectrom Rev 27(1):1–19CrossRefPubMedGoogle Scholar
  17. Fraterman S, Zeiger U, Khurana TS, Rubinstein NA, Wilm M (2007) Combination of peptide OFFGEL fractionation and label-free quantitation facilitated proteomics profiling of extraocular muscle. Proteomics 7(18):3404–3416CrossRefPubMedGoogle Scholar
  18. Gauci S, van Breukelen B, Lemeer SM, Krijgsveld J, Heck AJ (2008) A versatile peptide pI calculator for phosphorylated and N-terminal acetylated peptides experimentally tested using peptide isoelectric focusing. Proteomics 8(23–24):4898–4906CrossRefPubMedGoogle Scholar
  19. Gilar M, Jaworski A, Olivova P, Gebler JC (2007) Peptide retention prediction applied to proteomic data analysis. Rapid Commun Mass Spectrom 21(17):2813–2821CrossRefPubMedGoogle Scholar
  20. Gupta N, Tanner S, Jaitly N, Adkins JN, Lipton M, Edwards R, Romine M, Osterman A, Bafna V, Smith RD, Pevzner PA (2007) Whole proteome analysis of post-translational modifications: Applications of mass-spectrometry for proteogenomic annotation. Genome Res 17(9):1362–1377CrossRefPubMedGoogle Scholar
  21. Heller M, Michel PE, Morier P, Crettaz D, Wenz C, Tissot JD, Reymond F, Rossier JS (2005a) Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma. Electrophoresis 26(6):1174–1188CrossRefPubMedGoogle Scholar
  22. Heller M, Ye M, Michel PE, Morier P, Stalder D, Junger MA, Aebersold R, Reymond F, Rossier JS (2005b) Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides. J Proteome Res 4(6):2273–2282CrossRefPubMedGoogle Scholar
  23. Horth P, Miller CA, Preckel T, Wenz C (2006) Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis  10.1074/mcp.T600037-mcp200. Mol Cell Proteomics 5(10):1968–1974Google Scholar
  24. Kay R, Barton C, Ratcliffe L, Matharoo-Ball B, Brown P, Roberts J, Teale P, Creaser C (2008) Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis. Rapid Commun Mass Spectrom 22(20):3255–3260CrossRefPubMedGoogle Scholar
  25. Kino K, Sugiyama H (2001) Possible cause of g–c→c–g transversion mutation by guanine oxidation product, imidazolone. Chem Biol 8(4):369–378CrossRefPubMedGoogle Scholar
  26. Krijgsveld J, Gauci S, Dormeyer W, Heck AJ (2006) In-gel isoelectric focusing of peptides as a tool for improved protein identification. J Proteome Res 5(7):1721–1730CrossRefPubMedGoogle Scholar
  27. Krokhin OV (2006) Sequence-specific retention calculator. Algorithm for peptide retention prediction in ion-pair RP-HPLC: application to 300- and 100-a pore size c18 sorbents. Anal Chem 78(22):7785–7795CrossRefPubMedGoogle Scholar
  28. Krokhin OV, Craig R, Spicer V, Ens W, Standing KG, Beavis RC, Wilkins JA (2004) An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC: its application to protein peptide mapping by off-line HPLC-MALDI MS. Mol Cell Proteomics 3(9):908–919CrossRefPubMedGoogle Scholar
  29. Lam H, Aebersold R (2010) Spectral library searching for peptide identification via tandem MS. Methods Mol Biol 604:95–103CrossRefPubMedGoogle Scholar
  30. Lengqvist J, Uhlen K, Lehtio J (2007) iTRAQ compatibility of peptide immobilized pH gradient isoelectric focusing. Proteomics 7(11):1746–1752CrossRefPubMedGoogle Scholar
  31. Mant CT, Burke TW, Black JA, Hodges RS (1988) Effect of peptide chain length on peptide retention behaviour in reversed-phase chromatography. J Chromatogr 458:193–205CrossRefPubMedGoogle Scholar
  32. Matthiesen R, Trelle MB, Hojrup P, Bunkenborg J, Jensen ON (2005) Vems 3.0: algorithms and computational tools for tandem mass spectrometry based identification of post-translational modifications in proteins. J Proteome Res 4(6):2338–2347CrossRefPubMedGoogle Scholar
  33. Meek JL (1980) Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition. Proc Natl Acad Sci USA 77(3):1632–1636CrossRefPubMedGoogle Scholar
  34. Nielsen ML, Savitski MM, Zubarev RA (2006) Extent of modifications in human proteome samples and their effect on dynamic range of analysis in shotgun proteomics. Mol Cell Proteomics 5(12):2384–2391CrossRefPubMedGoogle Scholar
  35. Norbeck AD, Monroe ME, Adkins JN, Anderson KK, Daly DS, Smith RD (2005) The utility of accurate mass and LC elution time information in the analysis of complex proteomes. J Am Soc Mass Spectrom 16(8):1239–1249CrossRefPubMedGoogle Scholar
  36. Olsen JV, Mann M (2004) Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation. Proc Natl Acad Sci USA 101(37):13417–13422CrossRefPubMedGoogle Scholar
  37. Savitski MM, Nielsen ML, Zubarev RA (2006) Modificomb, a new proteomic tool for mapping substoichiometric post-translational modifications, finding novel types of modifications, and fingerprinting complex protein mixtures. Mol Cell Proteomics 5(5):935–948CrossRefPubMedGoogle Scholar
  38. Shilov IV, Seymour SL, Patel AA, Loboda A, Tang WH, Keating SP, Hunter CL, Nuwaysir LM, Schaeffer DA (2007) The paragon algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra  10.1074/mcp.T600050-mcp200. Mol Cell Proteomics 6(9):1638–1655Google Scholar
  39. Spicer V, Yamchuk A, Cortens J, Sousa S, Ens W, Standing KG, Wilkins JA, Krokhin OV (2007) Sequence-specific retention calculator. A family of peptide retention time prediction algorithms in reversed-phase HPLC: applicability to various chromatographic conditions and columns. Anal Chem 79(22):8762–8768CrossRefPubMedGoogle Scholar
  40. Stephenson JL Jr, Bunger MK, Cargile BJ, Sevinsky JR (2006) A new algorithm for pi prediction of peptides from IPG-IEF: applications to analysis of single nucleotide polymorphisms. In: 7th Siena meeting from genome to proteome: back to the future, Siena, Italy, 3–7 September 2006Google Scholar
  41. Tanner S, Shu H, Frank A, Wang LC, Zandi E, Mumby M, Pevzner PA, Bafna V (2005) Inspect: identification of posttranslationally modified peptides from tandem mass spectra. Anal Chem 77(14):4626–4639CrossRefPubMedGoogle Scholar
  42. Uhlén K, Fenyo D, Hörnsten L, Bjellqvist B (2006) Improved prediction of peptide isoelectric point by modelling the effects of interaction between charged neighbouring amino acids. In: 7th Siena meeting from genome to proteome: back to the future, Siena, Italy, 3–7 September 2006Google Scholar
  43. Vaezzadeh AR, Hernandez C, Vadas O, Deshusses JJ, Lescuyer P, Lisacek F, Hochstrasser DF (2008) PICarver: a software tool and strategy for peptides isoelectric focusing. J Proteome Res 7(10):4336–4345CrossRefPubMedGoogle Scholar
  44. Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138(1):141–143CrossRefPubMedGoogle Scholar
  45. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362CrossRefPubMedGoogle Scholar
  46. Xie H, Gilar M, Gebler JC (2009) Characterization of protein impurities and site-specific modifications using peptide mapping with liquid chromatography and data independent acquisition mass spectrometry. Anal Chem 81(14):5699–5708CrossRefPubMedGoogle Scholar
  47. Zubarev R, Mann M (2007) On the proper use of mass accuracy in proteomics. Mol Cell Proteomics 6(3):377–381PubMedGoogle Scholar
  48. Zybailov B, Sun Q, van Wijk KJ (2009) Workflow for large scale detection and validation of peptide modifications by RPLC-LTQ-Orbitrap: application to the Arabidopsis thaliana leaf proteome and an online modified peptide library. Anal Chem 81(19):8015–8024CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Johan Lengqvist
    • 1
    • 2
  • Hanna Eriksson
    • 2
    • 4
  • Marcus Gry
    • 1
  • Kristina Uhlén
    • 5
  • Christina Björklund
    • 1
  • Bengt Bjellqvist
    • 5
  • Per-Johan Jakobsson
    • 2
    • 4
  • Janne Lehtiö
    • 2
    • 3
  1. 1.Department of Safety Assessment, Molecular ToxicologyAstraZeneca R&DSödertäljeSweden
  2. 2.Karolinska Biomics CenterKarolinska University HospitalStockholmSweden
  3. 3.Department of Oncology and Pathology, Karolinska Biomics Center (Z5:02)Karolinska InstitutetStockholmSweden
  4. 4.Rheumatology Unit, Department of MedicineKarolinska InstitutetStockholmSweden
  5. 5.GE Healthcare Bio-Sciences ABUppsalaSweden

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