SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle

Abstract

The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues.

Biological significance: Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results.

Keywords: Insulin signaling; Phosphoproteomics; Quantitative proteomics; Tissue proteomics.

Figure 1. Assessment of post-phosphopeptide enrichment chemical tagging

A, SILAC scheme. HeLa cells were metabolically labeled with light (orange) and heavy (green) amino acids in cell culture. Labeled cells were mixed, trypsin-digested, phosphopeptides were enriched using titanium dioxide microspheres, and analyzed by LC-MS/MS. B, Reductive dimethyl-labeling scheme. HeLa cells were trypsin digested, and phosphopeptides were enriched using titanium dioxide microspheres. Phosphopeptides were labeled separately using heavy and light reductive dimethyl-labeling chemistry, mixed, and analyzed by LC-MS/MS. C, Log₂ ratio distribution of biological (DM1-3) and technical (DMT1-3) replicates of phosphopeptides labeled by reductive dimethyl-labeling chemistry. D, Log₂ ratio distribution of biological replicates of phosphopeptides labeled by reductive dimethyl-labeling chemistry (DM1-3) and SILAC (SILAC1-3).

Figure 2. Application to insulin signaling in the murine myoblast cell line C2C12

A, Labeling scheme and log₂ ratio distribution of phosphopeptides from insulin versus control-treated C2C12 cells. C2C12 cells were metabolically labeled with light (orange) and heavy (green) amino acids in cell culture. For reductive dimethyl-labeling, C2C12 cells grown in light SILAC media were treated with insulin or not for 10 min. For SILAC labeling, heavy-labeled C2C12 cells were treated with insulin for 10 min, while light-labeled C2C12 cells were left untreated. B. Distribution of p-values as determined by Students T-Test of phosphopeptides identified in each of the triplicate SILAC or reductive dimethyl-labeling analysis. C, Pearson correlation analysis of log₂ ratios of phosphopeptides quantified in all six analyses with a p-value < 0.05 (Students T-Test).

Figure 3. Application to insulin signaling in murine skeletal muscle tissue

A, Log₂ ratio distribution of phosphopeptides from insulin versus control-treated mice. Total numbers of phosphopeptides identified in each experiment are indicated. B. Distribution of p-values (Students T-Test) of phosphopeptides quantified in the three analyses. C, Pearson correlation analysis of phosphopeptide log₂ ratios quantified in all three analysis with a p-value < 0.1 (Students T-Test) identified in all three experiments.

Figure 4. Validation of mass spectrometry results

Western blot analysis of important phosphorylation sites in insulin signaling in control and insulin-stimulated mice.

Figure 5. Bioinformatic analysis of insulin-regulated phosphorylation sites

Pathway analysis on protein complexes containing phosphorylation sites that were quantified with p-value < 0.05 (Students T-Test) using Pathway Studio software. Phosphoproteins identified in this study and their degrees of regulation are shown in color; other proteins in the pathway are shown in gray.