title: Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis.
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.
authors.affiliation: Department of Protein Technology, Lund University, Lund, Sweden.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Arrigoni
authors.forename: Svante
authors.initials: S
authors.lastname: Resjö
authors.forename: Fredrik
authors.initials: F
authors.lastname: Levander
authors.forename: Rebecka
authors.initials: R
authors.lastname: Nilsson
authors.forename: Eva
authors.initials: E
authors.lastname: Degerman
authors.forename: Manfredo
authors.initials: M
authors.lastname: Quadroni
authors.forename: Lorenzo A
authors.initials: LA
authors.lastname: Pinna
authors.forename: Peter
authors.initials: P
authors.lastname: James
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D000818:Animals; D002364:Caseins / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D047468:Immunoprecipitation; D008297:Male; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis*; D010769:Phosphothreonine / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002364:Caseins; D010748:Phosphopeptides; D010769:Phosphothreonine; D010768:Phosphoserine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3