Screening for Protein Phosphorylation Using Nanoscale Reactions on Microdroplet Arrays

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. Phosphopeptide screening: A mass spectrometry‐based screening method detects protein phosphorylation in complex protein mixtures without extensive MS/MS experiments. The method employs droplet microfluidics to integrate nanoliter phosphatase reactions in a nano‐LC‐MALDI‐MS workflow. The selective dephosphorylation of every second LC fraction induces characteristic peak fluctuations that can be used to identify even low‐abundant phosphopeptides.