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

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.

Figure 1.

TMT labeling increases MS1 single intensity and alters retention time. The synthetic QPSSSR library phosphopeptides with (red color) and without (blue color) TMT labeling were directly analyzed by LC-MS/MS without using a trapping column. (a) Representative MS1 signals.(b) Extracted ion chromatograms (XICs) for the QPSSSR phosphopeptide library with and without TMT0 labeling. RT, retention time.

Figure 2.

TMT labeling provides a greater shift in retention time for peptides containing lysine. (a) Retention time shift of phosphopeptides after TMT labeling (light blue, data points with the highest density; bright green, data points with the lowest density). (b) Hydrophobicity distribution of identified phosphopeptides. (c) Median retention time shift of phosphopeptides with or without lysine (K) residues. (d) Peak area ratio of phosphopeptides before and after TMT labeling. (e) Schematic diagram of increasing peptide hydrophobicity with TMT labeling.

Figure 3.

Endogenous detection of singly phosphorylated QPSSSR peptides. (a) Workflow for proteomic analysis of immunoprecipitated CXCR3 samples. Evidence for detection of endogenous singly phosphorylated QPSSSR peptides at (b) the MS1 level and (c) the MS2 level. The Δm/z of5.004 is the m/z difference between endogenous and synthesized heavy isotopic peptides. Neutral loss of phosphate group with a reduction of 98 Da in mass is very common for MS/MS sequencing of phosphopeptides, and thus, it was used as a signature for phosphopeptide identification.

Figure 4.

Quantification of endogenous QPSSSR peptides. QPSSSR phosphopeptides obtained through immunoprecipitation and tryptic digestion of CXCR3 were referenced to spiked-in as heavy QPSSSR library standards. (a) XICs of fragment ions. (b) Equation utilized for estimating phosphorylation stoichiometry of endogenous QPSSSR. (c) Estimated phosphorylation stoichiometry for singly phosphorylated QPSSSR peptides for samples treated either with vehicle or CXCL10. Error bars indicate mean ± SD. The p-value calculated with t test was 0.03.