Screening for Protein Phosphorylation Using Nanoscale Reactions on Microdroplet Arrays |
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Authors: | Dr Martin Pabst Prof Renato Zenobi Prof Petra S Dittrich |
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Institution: | 1. Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir‐Prelog‐Weg 3, 8093 Zurich (Switzerland)These authors contributed equally to this work and share first authorship.;2. Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir‐Prelog‐Weg 3, 8093 Zurich (Switzerland);3. Current address: Department of Biosystems Science and Engineering, ETH Zurich (Switzerland) |
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Abstract: | 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. |
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Keywords: | droplet microfluidics mass spectrometry microarrays protein modifications protein phosphorylation |
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