Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome.

ABSTRACT: Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we i

SUBMITTER: Panizza E 

PROVIDER: S-EPMC5495806 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature