Skip to main content
release_alert
Welcome to the new Scholars 3.0! Read about new features and let us know what you think.
cancel

Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides.

Publication ,  Journal Article
Tsai, C-F; Smith, JS; Eiger, DS; Martin, K; Liu, T; Smith, RD; Shi, T; Rajagopal, S; Jacobs, JM
Published in: Methods Mol Biol
2021

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.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

Methods Mol Biol

DOI

EISSN

1940-6029

Publication Date

2021

Volume

2259

Start / End Page

247 / 257

Location

United States

Related Subject Headings

  • Tandem Mass Spectrometry
  • Proteomics
  • Proteome
  • Phosphopeptides
  • MCF-7 Cells
  • Immunoprecipitation
  • Hydrophobic and Hydrophilic Interactions
  • Humans
  • HEK293 Cells
  • Developmental Biology
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Tsai, C.-F., Smith, J. S., Eiger, D. S., Martin, K., Liu, T., Smith, R. D., … Jacobs, J. M. (2021). Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides. Methods Mol Biol, 2259, 247–257. https://doi.org/10.1007/978-1-0716-1178-4_16
Tsai, Chia-Feng, Jeffrey S. Smith, Dylan S. Eiger, Kendall Martin, Tao Liu, Richard D. Smith, Tujin Shi, Sudarshan Rajagopal, and Jon M. Jacobs. “Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides.Methods Mol Biol 2259 (2021): 247–57. https://doi.org/10.1007/978-1-0716-1178-4_16.
Tsai C-F, Smith JS, Eiger DS, Martin K, Liu T, Smith RD, et al. Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides. Methods Mol Biol. 2021;2259:247–57.
Tsai, Chia-Feng, et al. “Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides.Methods Mol Biol, vol. 2259, 2021, pp. 247–57. Pubmed, doi:10.1007/978-1-0716-1178-4_16.
Tsai C-F, Smith JS, Eiger DS, Martin K, Liu T, Smith RD, Shi T, Rajagopal S, Jacobs JM. Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides. Methods Mol Biol. 2021;2259:247–257.

Published In

Methods Mol Biol

DOI

EISSN

1940-6029

Publication Date

2021

Volume

2259

Start / End Page

247 / 257

Location

United States

Related Subject Headings

  • Tandem Mass Spectrometry
  • Proteomics
  • Proteome
  • Phosphopeptides
  • MCF-7 Cells
  • Immunoprecipitation
  • Hydrophobic and Hydrophilic Interactions
  • Humans
  • HEK293 Cells
  • Developmental Biology