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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

Abstract

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.

Keywords

Isoelectric focusing Post-translational modifications Retention time 

Notes

Acknowledgments

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)

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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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