Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Abstract

Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

“…However, running sequential scans undoubtedly lowers instrument duty cycle and therefore sensitivity and sequence coverage, especially with ETD scans consuming milliseconds [51, 54]. In addition, it often requires tedious sample preparation and complex data processing since a complete picture is divided and distributed among several spectra [31, 55]. To address these existing limitations, this study aims to employ a hybrid EThcD approach to characterize intact glycopeptides in a one-spectrum fashion with much improved instrument duty cycle.…”

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

“…With this approach, we were able to substantially increase our knowledge of the native human O-glycoproteome by unambiguously identifying 1123 sites on 649 proteins, including identification of 541 sites and 231 O-glycoproteins that had not previously been found in native samples. 30,57,60 It should be noted, however, that these data likely underestimate the total number of O-glycans present because of the inherent limitations of shotgun proteomics. 27 This is of particular concern in regards to mucin-like repeat regions and highly homologous protein families that are refractory to mapping by mass spectrometry.…”

“…These subpatterns could suggest discrete roles for O-glycans worthy of further investigation; however, such manual analysis is biased by the types of proteins studied, prior knowledge, and potential effects of nonrandom Ser/Thr distribution. Therefore, we performed a systematic and unbiased association study using the full complement of protein annotations available from UniprotKB 54 and all O-glycosites described to date 1,8,[28][29][30]32,47,[55][56][57] (supplemental Table 1). This analysis identifies whether O-glycosites are over-or underenriched around (6 15 amino acids) each protein annotation relative to the expected frequency based on the background distribution of Ser/Thr residues, and is somewhat analogous to a gene ontology study, in which gene ontologies are replaced with protein annotations (Figure 6A).…”

“…Recent developments in mass spectrometry and glycan-enrichment methods, however, have enabled global studies of site-specific glycosylation using native tissues. 8,[27][28][29][30] Such glycoproteomic analyses have been applied to identify O-glycosites in human cerebrospinal fluid 28 and urine. 29 More recently, substantial progress has been made in the analysis of human plasma and serum, 30,31 although the overall number of glycopeptides identified has been modest because of difficulties achieving high-sensitivity glycopeptide enrichment.…”

Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides

“…A human serum-specific N-linked peptide database that was generated using solid phase extraction of glycosite-containing peptides (SPEG)1137 and solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG)18 in previous studies was used. The O-linked peptide database was also from previous studies3839. A minimum of two oxonium ions in top 10% of peaks in the spectra was required in an MS/MS spectrum to qualify for glycopeptide search.…”