Parallel Comparison of N‑Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC

Protein glycosylation is
of increasing interest due to its important roles in protein function
and aberrant expression with disease. Characterizing protein glycosylation
remains analytically challenging due to its low abundance, ion suppression
issues, and microheterogeneity at glycosylation sites, especially
in complex samples such as human plasma. In this study, the utility
of three common N-linked glycopeptide enrichment techniques is compared
using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer,
electrostatic repulsion hydrophilic interaction liquid chromatography
using strong anion exchange solid-phase extraction (SAX-ERLIC) provided
the most extensive N-linked glycopeptide enrichment when compared
with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC
enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across
72 glycosylation sites from 48 glycoproteins, which is more than double
that detected using other enrichment techniques. The greatest glycoform
diversity was observed in SAX-ERLIC enrichment, with no apparent bias
toward specific glycan types. SAX-ERLIC enrichments were additionally
analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize
glycopeptide identifications for a more comprehensive assessment of
protein glycosylation. In these experiments, 829 unique glycoforms
were identified across 208 glycosylation sites from 95 plasma glycoproteins,
a significant improvement from the initial method comparison and one
of the most extensive site-specific glycosylation analysis in immunodepleted
human plasma to date. Data are available via ProteomeXchange with
identifier PXD005655.