Protein phosphorylation is one of the most important cellular events and, probably, the most flexible and potent way the cell uses to regulate its functions. Approximately 30% of all proteins in mammalian organisms are phosphorylated and protein kinases represent the largest known protein family in eukaryotic genomes. It has been calculated that serine phosphorylation accounts for about 90% of the total phosphorylation events, while threonine and tyrosine phosphorylations account respectively for about 10% and 0.1% of the total phosphorylation. We have optimized a new derivatization reaction for the analysis of Ser/Thr phosphorylation by mass spectrometry. The strategy involves a beta-elimination/Michael addition reaction using 4-mercaptoethylpyridine as nucleophile. The substitution of the negatively charged phosphate group with a new positively charged pyridyl group strongly increases the ionisation and the detection of phosphorylated peptides, both using MALDI and ESI sources. Our experiments show that the detection of the phosphopeptides is increased by 50-100 folds on average. Moreover the fragmentation of the modified peptides under CID conditions gives rise to a specific reporter ion that can be used for parent ion analysis. The analysis of bovine alpha casein after the modification reaction shows the efficacy of this new method: all the known phosphorylation sites were identified together with a new phosphorylated serine.

A new derivatization reaction for the analysis of Ser/Thr phosphorylation by mass spectrometry

ARRIGONI, GIORGIO
2004

Abstract

Protein phosphorylation is one of the most important cellular events and, probably, the most flexible and potent way the cell uses to regulate its functions. Approximately 30% of all proteins in mammalian organisms are phosphorylated and protein kinases represent the largest known protein family in eukaryotic genomes. It has been calculated that serine phosphorylation accounts for about 90% of the total phosphorylation events, while threonine and tyrosine phosphorylations account respectively for about 10% and 0.1% of the total phosphorylation. We have optimized a new derivatization reaction for the analysis of Ser/Thr phosphorylation by mass spectrometry. The strategy involves a beta-elimination/Michael addition reaction using 4-mercaptoethylpyridine as nucleophile. The substitution of the negatively charged phosphate group with a new positively charged pyridyl group strongly increases the ionisation and the detection of phosphorylated peptides, both using MALDI and ESI sources. Our experiments show that the detection of the phosphopeptides is increased by 50-100 folds on average. Moreover the fragmentation of the modified peptides under CID conditions gives rise to a specific reporter ion that can be used for parent ion analysis. The analysis of bovine alpha casein after the modification reaction shows the efficacy of this new method: all the known phosphorylation sites were identified together with a new phosphorylated serine.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/1365967
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