ZIC-cHILIC-Based StageTip for Simultaneous Glycopeptide Enrichment and Fractionation toward Large-Scale N‑Sialoglycoproteomics

Alterations of protein
glycosylation are closely related with pathophysiological
regulation. Due to the structural macro- and microheterogeneity, low
stoichiometry, and low ionization efficiency of glycopeptides, high-performance
tools to enrich glycopeptides, especially the negatively charged and
labile sialoglycopeptides, are essential to enhance the identification
of the underexplored glycoproteome. Here, we present the first implementation
of zwitterionic hydrophilic interaction chromatography with the exposed
choline group (ZIC-cHILIC) in StageTip for simultaneous enrichment
and fractionation of intact glycopeptides. In a model study using
lung cancer cells, early elution by a high percentage of acetonitrile
prominently prefilters nonglycopeptides, facilitating high enrichment
specificity for glycopeptides (92–96%) and sialoglycopeptides
(77–89%) in the subsequent hydrophilic fractions. The stepwise
elution shows a high glycopeptide fractionation efficiency by a <10%
overlap of glycopeptides between adjacent fractions. Most importantly,
the ZIC-cHILIC stepwise strategy demonstrated good reproducibility
(>80% in triplicate analysis) as well as superior coverage of 4.6-
to 12.0-fold and 2.1- to 35.6-fold more glycopeptides and sialoglycopeptides
compared to conventional TiO2 and ZIC-HILIC, respectively.
To the best of our knowledge, the result with 2742 sialoglycopeptides
among 7367 unique glycopeptides and 166 glycans from 2434 N-glycosites
of 1118 glycoproteins (Byonic score > 100) provides one of the
deepest
glycoproteomic profiles in single-cell type. Without the immunoprecipitation
step, the large-scale glycoproteomic atlas also reveals site-specific
glycosylation of many druggable receptor proteins, such as EGFR, MET,
ERBB2, ERBB3, AXL, and IGF1R. The demonstrated high enrichment specificity
and identification depth show that stepwise ZIC-cHILIC is an efficient
method to explore the under-represented sialoglycoproteome.