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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

Arrigoni, Giorgio LU ; Resjö, Svante LU ; Levander, Fredrik LU ; Nilsson, R; Degerman, Eva LU ; Quadroni, M; Pinna, LA and James, Peter LU (2006) In Proteomics 6(3). p.757-766
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
matrix-assisted laser desorption/ionization time of, derivatization, flight mass spectrometry, phosphorylation, sensitivity, spectrometry, tandem mass
in
Proteomics
volume
6
issue
3
pages
757 - 766
publisher
John Wiley & Sons
external identifiers
  • pmid:16372258
  • wos:000235414600003
  • scopus:32944464615
ISSN
1615-9861
DOI
10.1002/pmic.200500073
language
English
LU publication?
yes
id
921ec828-a57b-4bf5-abc1-f8f519bda4f5 (old id 417345)
date added to LUP
2007-09-25 11:23:12
date last changed
2019-03-05 01:59:21
@article{921ec828-a57b-4bf5-abc1-f8f519bda4f5,
  abstract     = {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.},
  author       = {Arrigoni, Giorgio and Resjö, Svante and Levander, Fredrik and Nilsson, R and Degerman, Eva and Quadroni, M and Pinna, LA and James, Peter},
  issn         = {1615-9861},
  keyword      = {matrix-assisted laser desorption/ionization time of,derivatization,flight mass spectrometry,phosphorylation,sensitivity,spectrometry,tandem mass},
  language     = {eng},
  number       = {3},
  pages        = {757--766},
  publisher    = {John Wiley & Sons},
  series       = {Proteomics},
  title        = {Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis},
  url          = {http://dx.doi.org/10.1002/pmic.200500073},
  volume       = {6},
  year         = {2006},
}