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

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.