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
Language
- eng
Conference Location
- United States