Offline High pH Reversed-Phase Peptide Fractionation for Deep Phosphoproteome Coverage

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Abstract

Protein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental and metabolic stimuli, and are thus highly regulated. Therefore, studying the mechanism of regulation by phosphorylation, and pinpointing the exact site of phosphorylation on proteins is of high importance. This protocol describes in detail a phosphoproteomics workflow for ultra-deep coverage by fractionating peptide mixtures based on high pH (basic) reversed-phase chromatography prior to phosphopeptide enrichment and mass spectrometric analysis. Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions. Each Fraction is subsequently enriched for phosphopeptides using TiO2 followed by LC/MS analysis.

OriginalsprogEngelsk
TitelPhospho-Proteomics : Methods and Protocols
RedaktørerLouise von Stechow
Antal sider14
Vol/bind1355
ForlagSpringer
Publikationsdato2016
Sider179-92
ISBN (Trykt)978-1-4939-3048-7
ISBN (Elektronisk)978-1-4939-3049-4
DOI
StatusUdgivet - 2016
NavnMethods in molecular biology (Clifton, N.J.)
ISSN1064-3745

Bibliografisk note

AR2016

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