title: Screening for protein phosphorylation using nanoscale reactions on microdroplet arrays.
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.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich (Switzerland).
authors.forename: Simon K
authors.initials: SK
authors.lastname: Küster
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
authors.forename: Petra S
authors.initials: PS
authors.lastname: Dittrich
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D036103:Nanotechnology; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010748:Phosphopeptides; D011506:Proteins; D000469:Alkaline Phosphatase
keywords: droplet microfluidics; mass spectrometry; microarrays; protein modifications; protein phosphorylation