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Anal Chem. 2019 Sep 17;91(18):11606-11613. doi: 10.1021/acs.analchem.9b01814. Epub 2019 Aug 28.

Tandem Mass Tag Labeling Facilitates Reversed-Phase Liquid Chromatography-Mass Spectrometry Analysis of Hydrophilic Phosphopeptides.

Author information

1
Biological Sciences Division , Pacific Northwest National Laboratory , Richland , Washington 99354 , United States.
2
Department of Biochemistry , Duke University , Durham , North Carolina 27710 , United States.
3
Department of Medicine , Duke University , Durham , North Carolina 27710 , United States.
4
Department of Biochemistry and Biophysics , University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 , United States.
5
Environmental Molecular Sciences Laboratory , Pacific Northwest National Laboratory , Richland , Washington 99354 , United States.

Abstract

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. 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. Different from conventional TMT labeling, this method capitalizes on using a nonprimary amine buffer and TMT labeling occurring before C18-based solid phase extraction. Through phosphoproteomic analyses of MCF7 cells, we have demonstrated that this method can greatly increase the number of identified hydrophilic phosphopeptides and improve MS detection signals. We applied this method to study the peptide QPSSSR, a very hydrophilic tryptic peptide located on the C-terminus of the G protein-coupled receptor (GPCR) CXCR3. Identification of QPSSSR has never been reported, and we were unable to detect it by traditional methods. We validated our TMT labeling strategy by comparative RPLC-MS analyses of both a hydrophilic QPSSSR peptide library as well as common phosphopeptides. We further confirmed the utility of this method by quantifying QPSSSR phosphorylation abundances in HEK 293 cells under different treatment conditions predicted to alter QPSSSR phosphorylation. We anticipate that this simple TMT labeling method can be broadly used not only for decoding GPCR phosphoproteome but also for effective RPLC-MS analysis of other highly hydrophilic analytes.

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