A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: Chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry

Florian Thaler, Barbara Valsasina, Rosario Baldi, Jin Xie, Albert Stewart, Antonella Isacchi, Henryk M. Kalisz, Luisa Rusconi

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

β-Elimination of the phosphate group on phosphoserine and phosphothreonine residues and addition of an alkyldithiol is a useful tool for analysis of the phosphorylation states of proteins and peptides. We have explored the influence of several conditions on the efficiency of this PO 4 3- elimination reaction upon addition of propanedithiol. In addition to the described influence of different bases, the solvent composition was also found to have a major effect on the yield of the reaction. In particular, an increase in the percentage of DMSO enhances the conversion rate, whereas a higher amount of protic polar solvents, such as water or isopropanol, induces the opposite effect. We have also developed a protocol for enrichment of the modified peptides, which is based on solid-phase covalent capture/release with a dithiopyridino-resin. The procedure for β-elimination and isolation of phosphorylated peptides by solid-phase capture/release was developed with commercially available α-casein. Enriched peptide fragments were characterized by MALDI-TOF mass spectrometric analysis before and after alkylation with iodoacetamide, which allowed rapid confirmation of the purposely introduced thiol moiety. Sensitivity studies, carried out in order to determine the detection limit, demonstrated that samples could be detected even in the low picomolar range by mass spectrometry. The developed solid-phase enrichment procedure based on reversible covalent binding of the modified peptides is more effective and significantly simpler than methods based on the interaction between biotin and avidin, which require additional steps such as tagging the modified peptides and work-up of the samples prior to the affinity capture step.

Original languageEnglish
Pages (from-to)366-373
Number of pages8
JournalAnalytical and Bioanalytical Chemistry
Volume376
Issue number3
DOIs
Publication statusPublished - Jun 2003

Fingerprint

Phosphothreonine
Phosphoserine
Chemical modification
Mass spectrometry
Mass Spectrometry
Peptides
Proteins
Iodoacetamide
Addition reactions
Phosphorylation
Peptide Fragments
2-Propanol
Avidin
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Alkylation
Base Composition
Biotin
Caseins
Dimethyl Sulfoxide
Sulfhydryl Compounds

Keywords

  • Beta-elimination of phosphate
  • Dithiopyridino-resin
  • MALDI
  • Mass spectrometry
  • Protein chemistry
  • Protein phosphorylation

ASJC Scopus subject areas

  • Analytical Chemistry
  • Clinical Biochemistry

Cite this

A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins : Chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry. / Thaler, Florian; Valsasina, Barbara; Baldi, Rosario; Xie, Jin; Stewart, Albert; Isacchi, Antonella; Kalisz, Henryk M.; Rusconi, Luisa.

In: Analytical and Bioanalytical Chemistry, Vol. 376, No. 3, 06.2003, p. 366-373.

Research output: Contribution to journalArticle

Thaler, Florian ; Valsasina, Barbara ; Baldi, Rosario ; Xie, Jin ; Stewart, Albert ; Isacchi, Antonella ; Kalisz, Henryk M. ; Rusconi, Luisa. / A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins : Chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry. In: Analytical and Bioanalytical Chemistry. 2003 ; Vol. 376, No. 3. pp. 366-373.
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