Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides.

Journal Article (Journal Article)

Protein phosphorylation is a critical posttranslational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein, we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.

Full Text

Duke Authors

Cited Authors

  • Tsai, C-F; Smith, JS; Eiger, DS; Martin, K; Liu, T; Smith, RD; Shi, T; Rajagopal, S; Jacobs, JM

Published Date

  • 2021

Published In

Volume / Issue

  • 2259 /

Start / End Page

  • 247 - 257

PubMed ID

  • 33687720

Pubmed Central ID

  • PMC8071202

Electronic International Standard Serial Number (EISSN)

  • 1940-6029

Digital Object Identifier (DOI)

  • 10.1007/978-1-0716-1178-4_16


  • eng

Conference Location

  • United States