Improving Blood Plasma Glycoproteome Coverage by Coupling Ultracentrifugation Fractionation to Electrostatic Repulsion–Hydrophilic Interaction Chromatography Enrichment

Blood
plasma is considered to be an excellent source of disease
biomarkers because it contains proteins, lipids, metabolites, cell,
and cell-derived extracellular vesicles from different cellular origins
including diseased tissues. Most secretory and membranous proteins
that can be found in plasma are glycoproteins; therefore, the plasma
glycoproteome is one of the major subproteomes that is highly enriched
with disease biomarkers. As a result, the glycoproteome has attracted
much attention in clinical proteomic research. The modification of
proteins with glycans regulates a wide range of functions in biology,
but profiling plasma glycoproteins on a global scale has been hampered
by the presence of low stoichiometry of glycoproteins in a complex
high abundance plasma proteome background and lack of effective analytical
technique. This study aims to improve plasma glycoproteome coverage
using pig plasma as a model sample with a two-step strategy. The first
step involves fractionation of the plasma proteins using ultracentrifugation
into supernatant and pellet that is believed to contain low abundant
glycoproteins. In the second step, further enrichment of glycopeptides
was achieved in both fractions by adopting electrostatic repulsion
hydrophilic interaction chromatography (ERLIC) coupled to tandem mass
spectrometry (LC–MS/MS) analysis. The coverage of enriched
glycoproteins in supernatant, pellet, and whole plasma sample as control
was compared. Using this simple sample fractionation approach by ultracentrifugation
and further ERLIC enrichment technique, sample complexity was reduced
and glycoproteome coverage was significantly enhanced in supernatant
and pellet fractions (by >50%) compared with whole plasma sample.
This study showed that when ultracentrifugation is coupled to ERLIC
glycopeptides enrichment and glycoproteome identification are significantly
improved. This study demonstrates the combination of ultracentrifugation
and ERLIC as a useful method for discovering plasma glycoprotein disease
biomarkers.