In-Depth Mass Spectrometry Analysis Reveals the Plasma Proteomic and N-Glycoproteomic Impact of an Amish-Enriched Cardioprotective Variant in B4GALT1

B4GALT1 encodes β-1,4-galactosyltransferase 1, an enzyme that plays a major role in glycan synthesis in the Golgi apparatus by catalyzing the addition of terminal galactose. Studies increasingly suggest that B4GALT1 may be involved in the regulation of lipid metabolism pathways. Recently, we discovered a single-site missense variant Asn352Ser (N352S) in the functional domain of B4GALT1 in an Amish population, which decreases the level of LDL-cholesterol (LDL-c) as well as the protein levels of ApoB, fibrinogen, and IgG in the blood. To systematically evaluate the effects of this missense variant on protein glycosylation, expression, and secretion, we developed a nano-LC-MS/MS-based platform combined with TMT-labeling for in-depth quantitative proteomic and glycoproteomic analyses in the plasma of individuals homozygous for the B4GALT1 missense variant N352S versus non-carriers (n = 5 per genotype). A total of 488 secreted proteins in the plasma were identified and quantified, 34 of which showed significant fold changes in protein levels between N352S homozygotes and non-carriers. We determined N-glycosylation profiles from 370 glycosylation sites in 151 glycoproteins and identified ten proteins most significantly associated with decreased galactosylation and sialyation in B4GALT1 N352S homozygotes. These results further support that B4GALT1 N352S alters the glycosylation profiles of a variety of critical target proteins, thus governing the functions of these proteins in multiple pathways, such as those involved in lipid metabolism, coagulation, and the immune response.