Tandem Mass Tag Labeling Facilitates Reversed-Phase Liquid Chromatography-Mass Spectrometry Analysis of Hydrophilic Phosphopeptides
- BATTELLE (PACIFIC NW LAB)
- Duke University
- University of North Carolina at Chapel Hill
Protein phosphorylation is a critical post-translational modification (PTM). 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 with enriched regions of serines, threonines, and tyrosines that often orchestrate critical biological functions. It is known that G protein coupled receptors (GPCRs) are regulators of cellular signaling events and rely on phosphorylation of serines and threonines within the GPCR C-terminus for appropriate signaling. However, the low abundance of GPCRs, combined with the hydrophilicity of C-terminal phosphopeptides, has prevented robust RPLC-MS analysis of potentially crucial regions. To address this issue, we developed a simple, easily implemented method to introduce a commonly used tandem mass tag (TMT) to increase peptide hydrophobicity, effectively enhancing RPLC-MS analysis of hydrophilic peptides. This method was applied to study the peptide QPSSSR, a tryptic peptide located on the C-terminus of the GPCR CXCR3 not been reported, and validated by comparative RPLC-MS analyses of both a hydrophilic QPSSSR peptide library as well as common phosphopeptides before and after TMT labeling. We further confirmed the utility of this method by quantifying QPSSSR phosphorylation abundances in HEK 293 cells under different treatement conditions predicted to alter QPSSSR phosphorylation. We anticipate that this simple TMT-labeling method can be broadly used not only for decoding GPCR phosphoproteomes, but also for effective RPLC-MS analysis of other highly hydrophilic analytes.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1577060
- Report Number(s):
- PNNL-SA-142808
- Journal Information:
- Analytical Chemistry, Vol. 91, Issue 18
- Country of Publication:
- United States
- Language:
- English
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