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Screening for Protein Phosphorylation Using Nanoscale Reactions on Microdroplet Arrays
Author(s) -
Küster Simon K.,
Pabst Martin,
Zenobi Renato,
Dittrich Petra S.
Publication year - 2015
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201409440
Subject(s) - chromatography , chemistry , phosphatase , alkaline phosphatase , elution , microarray , phosphorylation , enzyme , nanoscopic scale , protein microarray , mass spectrometry , biochemistry , nanotechnology , materials science , gene expression , gene
We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano‐LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n ×−80 Da. The MALDI‐MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions.