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Journal of The American Society for Mass Spectrometry

, Volume 30, Issue 9, pp 1578–1585 | Cite as

Electron Transfer/Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations

  • Martin PenkertEmail author
  • Anett Hauser
  • Robert Harmel
  • Dorothea Fiedler
  • Christian P. R. Hackenberger
  • Eberhard Krause
Research Article

Abstract

In recent years, labile phosphorylation sites on arginine, histidine, cysteine, and lysine as well as pyrophosphorylation of serine and threonine have gained more attention in phosphoproteomic studies. However, the analysis of these delicate posttranslational modifications via tandem mass spectrometry remains a challenge. Common fragmentation techniques such as collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) are limited due to extensive phosphate-related neutral loss. Electron transfer dissociation (ETD) has shown to preserve labile modifications, but is restricted to higher charge states, missing the most prevalent doubly charged peptides. Here, we report the ability of electron transfer/higher energy collisional dissociation (EThcD) to fragment doubly charged phosphorylated peptides without losing the labile modifications. Using synthetic peptides that contain phosphorylated arginine, histidine, cysteine, and lysine as well as pyrophosphorylated serine residues, we evaluated the optimal fragmentation conditions, demonstrating that EThcD is the method of choice for unambiguous assignment of tryptic, labile phosphorylated peptides.

Graphical Abstract

Keywords

Electron transfer/higher energy collisional dissociation EThcD Labile phosphorylation, doubly charged peptide ions Histidine phosphorylation Lysine phosphorylation Cysteine phosphorylation Arginine phosphorylation Pyrophosphorylation 

Notes

Supporting Information

Supplemental material, including Supplemental Tables TS1-TS14 and Supplemental Figures S1–S9 are available with this manuscript. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [45] partner repository with the dataset identifier PXD012989.

Supplementary material

13361_2019_2240_MOESM1_ESM.docx (2.2 mb)
ESM 1 (DOCX 2286 kb)

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Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)BerlinGermany
  2. 2.Department of ChemistryHumboldt-Universität zu BerlinBerlinGermany

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