Analysis of Viral Spike Protein N-Glycosylation Using Ultraviolet Photodissociation Mass Spectrometry

Abstract

Characterization of protein glycosylation by tandem mass spectrometry remains challenging owing to the vast diversity of oligosaccharides bound to proteins, the variation in monosaccharide linkage patterns, and the lability of the linkage between the glycan and protein. Here, we have adapted an HCD-triggered-ultraviolet photodissociation (UVPD) approach for the simultaneous localization of glycosites and full characterization of both glycan compositions and intersaccharide linkages, the latter provided by extensive cross-ring cleavages enabled by UVPD. The method is applied to study glycan compositions based on analysis of glycopeptides from proteolytic digestion of recombinant human coronaviruse spike proteins from SARS-CoV-2 and HKU1. UVPD reveals unique intersaccharide linkage information and is leveraged to localize N-linked glycoforms with confidence.

In Situ Imaging of O-Linked β-N-Acetylglucosamine Using On-Tissue Hydrolysis and MALDI Mass Spectrometry

“…As a first step, we evaluated the efficacy of on-slide hydrolysis of O-GlcNAc with Bacteroides thetaiotamicron β-N-acetylglucosaminidase (BtOGA), a bacterial OGA homologue from glycoside hydrolase family GH84 that cleaves O-GlcNAc [59] using a standard glycopeptide, O-GlcNAc-modified TAB1 389−401 (biotin-PVSVPYS395SAQSTS) [58,60]. To optimize enzymatic reaction conditions, we sprayed BtOGA onto dried spots of the TAB1 glycopeptide on glass slides.…”

“…A variety of MS/MS methods, including higher-energy collision dissociation (HCD), electron transfer dissociation (ETD), hybrid methods such as the combination of ETD with supplementary collisional energy (EThcD), and photon-based ultraviolet dissociation (UVPD), have proven successful. Each MS/MS method provides amino acid and glycan sequence information; however, EThcD and UVPD stand out by providing complete glycopeptide characterization [56][57][58].…”

Ultraviolet Photodissociation Permits Comprehensive Characterization of O-Glycopeptides Cleaved with O-Glycoprotease IMPa

“…38,39,43 For the present study, we have coupled the use of Oglycoprotease IMPa to generate O-glycopeptides and HCDtriggered UVPD to characterize the resulting O-glycopeptides and to pinpoint glycosite heterogeneity. 38 Performance metrics are compared for UVPD, HCD, and EThcD, with a focus on glycoproteins containing core 1 and core 2 O-glycans owing to their prevalence in biotherapeutics. We also evaluate the capability of UVPD to determine site heterogeneity among proximal and adjacent O-glycosites.…”

“…40,42 UVPD has also been used for the characterization of N-glycosylation sites. 38,39,43 For the present study, we have coupled the use of Oglycoprotease IMPa to generate O-glycopeptides and HCDtriggered UVPD to characterize the resulting O-glycopeptides and to pinpoint glycosite heterogeneity. 38 Performance metrics are compared for UVPD, HCD, and EThcD, with a focus on glycoproteins containing core 1 and core 2 O-glycans owing to their prevalence in biotherapeutics.…”

Higher Temperature Porous Graphitic Carbon Separations Differentially Impact Distinct Glycopeptide Classes

“…In order to offset the overwhelming difficulties in glycopeptide analysis, numerous areas of analytical development have received significant attention in recent years. Glycopeptide enrichment strategies have been developed to compensate for low analyte abundance in proteolytic mixtures, − with some being tailored to unique glycan classes. , In addition, ubiquitous utilization of tandem-MS for glycopeptide identification has brought about validation and application of numerous unique dissociation modalities. − Even efforts for accurate quantitation of intact glycopeptides have seen a rise in popularity as isotopic and isobaric labeling strategies become more accessible . These areas of development, however, largely ignore any potential benefits that may come through more effective chromatography.…”