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

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.

Phosphorylation barcodes direct biased chemokine signaling at CXCR3.

G protein-coupled receptor (GPCR) biased agonism, the activation of some signaling pathways over others, is thought to largely be due to differential receptor phosphorylation, or "phosphorylation barcodes." At chemokine receptors, ligands act as "biased agonists" with complex signaling profiles, which contributes to the limited success in pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of CXCR3 phosphosites altered ß-arrestin conformation in cellular assays and was confirmed by molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes and lead to distinct physiological processes.

Phosphorylation barcodes direct biased chemokine signaling at CXCR3.

G protein-coupled receptor (GPCR)-biased agonism, selective activation of certain signaling pathways relative to others, is thought to be directed by differential GPCR phosphorylation "barcodes." At chemokine receptors, endogenous chemokines can act as "biased agonists", which may contribute to the limited success when pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomics studies. Mutation of CXCR3 phosphosites altered ß-arrestin 2 conformation in cellular assays and was consistent with conformational changes observed in molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes, leading to distinct physiological processes.