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Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis

Author:
  • Giorgio Arrigoni
  • Svante Resjö
  • Fredrik Levander
  • R Nilsson
  • Eva Degerman
  • M Quadroni
  • LA Pinna
  • Peter James
Publishing year: 2006
Language: English
Pages: 757-766
Publication/Series: Proteomics
Volume: 6
Issue: 3
Document type: Journal article
Publisher: John Wiley & Sons

Abstract english

Protein phosphorylation is one of the most important and common ways of regulating protein function in cells. However, phosphopeptides are difficult to analyse, ionising poorly under standard MALDI conditions. Several methods have been developed to deal with the low sensitivity and specificity of phosphopeptide analysis. Here, we show an approach using a simple one-step beta-elimination/Michael addition reaction for the derivatization of phosphoserine and phosphothreonine. The substitution of the negatively charged phosphate group by a positively charged S-ethylpyridyl group greatly improves the ionisation of the modified peptides, especially in MALDI MS, increasing the sensitivity of the analysis. The modification allows the formation of a unique fragment ion at m/z 106 under mild collisional activation conditions, which can be used for parent (precursor) ion scanning in order to improve both the sensitivity and the selectivity of the analysis. The optimisation of the approach is described for a standard model peptide and protein and then applied to phosphorylation analysis in two biologically derived proteins purified from different experimental systems.

Keywords

  • Endocrinology and Diabetes
  • matrix-assisted laser desorption/ionization time of
  • derivatization
  • flight mass spectrometry
  • phosphorylation
  • sensitivity
  • spectrometry
  • tandem mass

Other

Published
  • Insulin Signal Transduction
  • ISSN: 1615-9861
Peter James
E-mail: peter.james [at] immun.lth.se

Professor

Department of Immunotechnology

+46 46 222 14 96

+46 70 247 79 60

MV406411E1

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