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

B4GALT1 encodes ß-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.

Benchmark of site- and structure-specific quantitative tissue N-glycoproteomics for discovery of potential N-glycoprotein markers: a case study of pancreatic cancer.

Pancreatic cancer is a highly malignant tumor of the digestive tract that is difficult to diagnose and treat. It is more common in developed countries and has become one of the main causes of death in some countries and regions. Currently, pancreatic cancer generally has a poor prognosis, partly due to the lack of symptoms in the early stages of pancreatic cancer. Therefore, most cases are diagnosed at advanced stage. With the continuous in-depth research of glycoproteomics in precision medical diagnosis, there have been some reports on quantitative analysis of cancer-related cells, plasma or tissues to find specific biomarkers for targeted therapy. This research is based on the developed complete N-linked glycopeptide database search engine GPSeeker, combined with liquid-mass spectrometry and stable diethyl isotope labeling, providing a benchmark of site- and structure-specific quantitative tissue N-glycoproteomics for discovery of potential N-glycoprotein markers. With spectrum-level FDR ≤1%, 20,038 intact N-Glycopeptides corresponding to 4518 peptide backbones, 228 N-glycan monosaccharide compositions 1026 N-glycan putative structures, 4460 N-glycosites and 3437 intact N-glycoproteins were identified. With the criteria of ≥1.5-fold change and p value<0.05, 52 differentially expressed intact N-glycopeptides (DEGPs) were found in pancreatic cancer tussues relative to control, where 38 up-regulated and 14 down-regulated, respectively.

Structural N-glycoproteomics characterization of cell-surface N-glycosylation of MCF-7/ADR cancer stem cells.

Breast cancer is responsible for the highest mortality all over the world. Cancer stem cells (CSCs) along with epithelial mesenchymal transition (EMT) are identified as a driver of cancer which are responsible for cancer metastasis and drug resistance. Several signaling pathways are associated with drug resistance. Additionally, glycosyltransferases regulate different types of glycosylation which are involved in drug resistance. To the end, it is urgent to figure out the knowledge on cell-surface altered N-glycosylation and putative markers. Here, differential cell-surface intact N-glycopeptides in adriamycin (ADR)-resistant michigan breast cancer foundation-7 stem cells (MCF-7/ADR CSCs) relative to ADR-sensitive MCF-7 CSCs were analyzed with site- and structure-specific quantitative N-glycoproteomics. The intact N-glycopeptides and differentially expressed intact N-glycopeptides (DEGPs) were determined and quantified via intact N-glycopeptide search engine GPSeeker. Totally, 4777 intact N-glycopeptides were identified and N-glycan sequence structures among 2764 IDs were distinguished from their isomers by structure-diagnostic fragment ions. Among 1717 quantified intact N-glycopeptides, 104 DEGPs were determined (fold change ≥ 1.5 and p value < 0.05). Annotation of protein-protein interaction and biological processes among others of DEGPs were finally carried out; down-regulated intact N-glycopeptide with bisecting GlcNAc from p38-interacting protein and up-regulated intact N-glycopeptide with ß1,6-branching N-glycan from integrin beta-5 were found.

Comparative glycoproteomics study on the surface of SKOV3 versus IOSE80 cell lines.

Objective: Site- and structure-specific quantitative N-glycoproteomics study of differential cell-surface N-glycosylation of ovarian cancer SKOV3 cells with the non-cancerous ovarian epithelial IOSE80 cells as the control. Methods: C18-RPLC-MS/MS (HCD with stepped normalized collision energies) was used to analyze the 1: 1 mixture of labeled intact N-glycopeptides from SKOV3 and IOSE80 cells, and the site- and structure-specific intact N-glycopeptide search engine GPSeeker was used to conduct qualitative and quantitative search on the obtained raw datasets. Results: With the control of the spectrum-level false discovery rate ≤1%, 13,822 glycopeptide spectral matches coming from 2,918 N-glycoproteins with comprehensive N-glycosite and N-glycan structure information were identified; 3,733 N-glycosites and 3,754 N-glycan sequence structures were confirmed by site-determining and structure-diagnostic fragment ions, respectively. With the control of no less than two observations among the three technical replicates, fold change ≥1.5, and p-value ≤ 0.05, 746 DEPGs in SKOV3 cells relative to IOSE80 cells were quantified, where 421 were upregulated and 325 downregulated. Conclusion: Differential cell-surface N-glycosylation of ovarian cancer SKOV3 cells were quantitatively analyzed by isotopic labeling and site- and structure-specific N-glycoproteomics. This discovery study provides putative N-glycoprotein biomarker candidates for future validation study using multiple reaction monitoring and biochemical methods.

Sialic acid linkage-specific quantitative N-glycoproteomics using selective alkylamidation and multiplex TMT-labeling.

Protein sialylation participates many biological processes in a linkage-specific manner, and aberrant sialylation has been associated with many malignant diseases. Mass spectrometry-based quantitative N-glycoproteomics has been widely adopted for quantitative analysis of aberrant sialylation, yet multiplexing method at intact N-glycopeptides level is still lacking. Here we report our study of sialic acid linkage-specific quantitative N-glycoproteomics using selective alkylamidation and multiplex tandem mass tags (TMT)-labeling. With lung cancer as a model system, differential sialylation in cancer tissues relative to adjacent non-tumor tissues was characterized at the intact N-glycopeptide level with N-glycosite information. TMT-labeled intact N-glycopeptides with and without sialic acid alkylamidation were subject to reversed-phase liquid chromatography-nano-electron spray ionization-tandem mass spectrometry (RPLC-nanoESI-MS/MS) analysis to provide comprehensive characterization of N-glycosylation with and without sialic acid at the intact N-glycopeptide level with structure and N-glycosite. In this study, 6384 intact N-glycopeptides without sialylation were identified and 521 differentially expressed intact N-glycopeptides from 254 intact N-glycoproteins were quantified. Eight intact N-glycoproteins responsible for N-glycan biosynthesis were identified as glycosyltransferases. In total, 307 sialylated intact N-glycopeptides with linkage-specific sialic acid residues were identified together with 29 N-glycans with α2,6-linked sialic acids and 55 N-glycans with α2,3-linked sialic acids. Intact N-glycoproteins with α2,6-sialylation were associated with coronavirus disease-(COVID)-19. Additionally, many types of N-glycosylation including terminal N-galactosylation, core and/or branch fucosylation, α2,6-sialylation and terminal bisecting N-acetylglucosamine were identified and quantified in intact N-glycoproteins from immunoglobulin family.