An Integrated Workflow for Global, Glyco-, and Phospho-proteomic Analysis of Tumor Tissues

Abstract

Recently, the rapid development and application of mass spectrometry (MS)-based technologies have markedly improved the comprehensive proteomic characterization of global proteome and protein post-translational modifications (PTMs). However, the current conventional approach for global proteomic analysis is often carried out separately from PTM analysis. In our study, we developed an integrated workflow for multiplex analysis of global, glyco-, and phospho-proteomics using breast cancer patient-derived xenograft (PDX) tumor samples. Our approach included the following steps: trypsin-digested tumor samples were enriched for phosphopeptides through immobilized metal ion affinity chromatography (IMAC), followed by enrichment of glycopeptides through mixed anion exchange (MAX) method, and then the flow-through peptides were analyzed for global proteomics. Our workflow demonstrated an increased identification of peptides and associated proteins in global proteome, as compared to those using the peptides without PTM depletion. In addition to global proteome, the workflow identified phosphopeptides and glycopeptides from the PTM enrichment. We also found a subset of glycans with unique distribution profiles in the IMAC flow-through, as compared to those enriched directly using the MAX method. Our integrated workflow provided an effective platform for simultaneous global proteomic and PTM analysis of biospecimens.

Figure 1.

Workflow for simultaneous analysis of global, phospho-, and glyco-proteomics. After protein extraction and trypsin digestion, the immobilized metal affinity chromatography (IMAC) was utilized to enrich phosphopeptides for phospho-proteomics analysis. Next, the flow-through from IMAC enrichment was enriched for intact glycopeptides (IGPs) using mixed anion exchange (MAX) cartridges. Finally, after depletion of glycopeptides and phosphopeptides, the remaining PTM-depleted global peptides (PTM-depleted Global) were used for global proteomics analysis. Tryptic-digested peptides without IMAC and MAX enrichment were directly analyzed for global proteomics for comparison (refer to as Global).

Figure 2.

Comparison of Global proteome with PTM-depleted Global proteome from the integrated workflow. (A) Number of PSMs in Global and PTM-depleted Global proteome. (B) Number of unique peptides in Global and PTM-depleted Global proteome. (C) Number of proteins in Global and PTM-depleted Global proteome. (D) Commonly identified proteins in Global and PTM-depleted Global proteome. (E) The proportion (%) of proteins using Gene Ontology (GO) molecular function assignment comparison between Global and PTM-depleted Global proteome. (F) Utilizing the spectral counting for protein quantification; the pie chart shows the distribution of PSMs over 100, 50–100, 20–50, 10–20, and less than 10 in Global and PTM-depleted Global.

Figure 3.

PTM analysis of sequential workflow. (A) The number of identified phosphopeptides, phospho-PSMs, and specificity in three replicates of IMAC enrichment. Specificity was calculated as phospho-PSMs/total PSMs. (B) Percentage of single, double, and triple phosphorylation events observed using IMAC enrichment. (C) Percentages of phospho-serine, -threonine, and -tyrosine identified in the IMAC enrichment process. (D) The number of identified intact glycopeptides from Global, PTM-depleted Global, IMAC, and MAX processes. (E) Percentages of different types of glycans identified in IGPs from the IMAC and MAX processes.

Figure 4.

Reproducibility of PTM-depleted Global. (A) Total of 1140 commonly identified proteins in three replicates. (B) Correlation matrix of the replicates generated based on 1140 common proteins. (C) Heatmap of Spearman correlation coefficients using protein spectral counting to show high reproducibility among three replicates with an average correlation coefficient >0.9. (D) Distribution of protein quantification based on spectral counting (log2 scale) in three replicates.

Figure 5.

Enzymatic removal of PTMs and identification results. (A) Workflow for global, PNGase F, and/or CIP-treated processes. (B) Number of protein identifications in Total Global, PNGaseF-treated global, CIP-treated global, and PNGaseF/CIP-treated global samples. (C) Peptide identifications in Total Global, PNGaseF-treated global, CIP-treated global, and PNGaseF/CIP-treated global proteomic analysis. (D) PSM identifications in Total Global, PNGase F-treated, CIP-treated, and PNGase F/CIP-treated global proteomic analysis.