Enrichment and nLC-MS/MS Analysis of Head and Neck Cancer Mucinome Glycoproteins

Mucin-domain
glycoproteins expressed on cancer cell surfaces play
central roles in cell adhesion, cancer progression, stem cell renewal,
and immune evasion. Despite abundant evidence that mucin-domain glycoproteins
are critical to the pathobiology of head and neck squamous cell carcinoma
(HNSCC), our knowledge of the composition of that mucinome is grossly
incomplete. Here, we utilized a catalytically inactive point mutant
of the enzyme StcE (StcEE447D) to capture mucin-domain
glycoproteins in head and neck cancer cell line lysates followed by
their characterization using sodium dodecyl-sulfate polyacrylamide
gel electrophoresis (SDS-PAGE), in-gel digestion, nano-liquid chromatography–tandem
mass spectrometry (nLC-MS/MS), and enrichment analyses. We demonstrate
the feasibility of this workflow for the study of mucin-domain glycoproteins
in HNSCC, identify a set of mucin-domain glycoproteins common to multiple
HNSCC cell lines, and report a subset of mucin-domain glycoproteins
that are uniquely expressed in HSC-3 cells, a cell line derived from
a highly aggressive metastatic tongue squamous cell carcinoma. This
effort represents the first attempt to identify mucin-domain glycoproteins
in HNSCC in an untargeted, unbiased analysis, paving the way for a
more comprehensive characterization of the mucinome components that
mediate aggressive tumor cell phenotypes. Data associated with this
study have been deposited to the ProteomeXchange Consortium via the
PRIDE partner repository with the data set identifier PXD029420.