Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows

Abstract

CD24Fc is a homodimeric recombinant Fc-fusion protein comprised of human CD24 connected to immunoglobulin G1 (IgG1) Fc fragment. CD24 is heavily glycosylated, and its biological function is considered mainly mediated by its glycosylation. Identification of the O-glycosylation sites would facilitate an in-depth understanding of the functional role of O-glycans in CD24. However, the presence of clustered mucin-type O-glycans together with N-glycans makes the determination of O-glycosylation sites and abundance very challenging. In this study, two sets of liquid chromatography-mass spectrometry (LC-MS) workflows were developed for the comprehensive characterization of O-glycosylation in CD24: (1) Fractionation and collision-induced dissociation (CID) workflow involving multienzyme digestion, fractionation, OpeRATOR/SialEXO digestion, and CID analysis; (2) Direct OpeRATOR/SialEXO digestion followed by electron-transfer/higher-energy collision dissociation (EThcD) analysis. The precise O-glycosylation sites were identified in CD24 for the first time, and the site occupancy was assessed. A total of 12 O-glycosylation sites were identified. Seven glycosylation sites were identified by both workflows, and five additional sites were identified only by the EThcD workflow. The predominant O-glycosylation site in CD24 was Thr25 followed by Thr15. The CID workflow provided an overall relative quantitation of O-glycoforms at the CD24 level and site localization for singly O-glycosylated peptides. The EThcD workflow directly identified glycosylation sites by tandem mass spectrometry (MS/MS) for singly, doubly, and triply O-glycosylated peptides. Together, both workflows validated each other’s results and can be applied to a complex mucin-type O-glycosylation site analysis of other glycoproteins and Fc-fusion therapeutics.

CD24 targeting with NK‐CAR immunotherapy in testis, prostate, renal and (luminal‐type) bladder cancer and identification of direct CD24 interaction partners

“…Here, analysis of PNGase treated chimera resolved in spectra with a high localization confidence. Additional, presumed O-glycan sites, without clear localization, were observed between L19 and S27 (T22/S26/S27) and between S42 and P49 (S42/ S44), again in line with previous reports [10]. The identified O-glycan compositions were HexNAc(1), Hex-NAc(1)Hex(1), HexNAc(1)Hex(1)NeuAc(1), and HexNAc(1)Hex(1)NeuAc(2) (Data S1B).…”

“…To comprehensively characterize the N ‐ and O ‐glycosylation pattern of CD24, liquid chromatography‐mass spectrometry (LC‐MS) analyses of a recombinant human CD24 Fc chimera expressed in mouse myeloma cells were performed. CD24 carries two N ‐glycosylation sequons at sites N36 and N52 [12], as well as two previously reported O ‐glycosylation sites at T41 and T51 [10]. Since CD24 is notoriously inaccessible to commonly used proteases such as trypsin, in the present study, we used a commercial recombinant CD24 chimera to test methods for detailed analysis and mapping of glycan microheterogeneity.…”

“…CD24 is a heavily glycosylated protein and our previous analyses demonstrated that glycosylation levels differed considerably between EC cell lines, prompting us to analyse the CD24 glycosylation in urological malignancies in more detail [4,10,11]. Enzymatic digestions of proteins isolated from 2102EP, NCCIT, NT2/D1, RT-112, PC-3 and Caki-1 cells utilizing PNGase F (cleaves N-glycans) and O-glycosidase/neuraminidase (cleaves O-glycans and sialic acid) revealed that, with the exception of PC-3, exclusively N-glycans were cleaved (Fig.…”

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

“…HCD and stepped HCD techniques have been used to study the expression of glycans on glycoproteins, determine glycan structures on viral proteins, and characterize the regulation of protein properties. − Stepped HCD methods (i.e., using multiple HCD collision energies) have revealed new O -glycosylation sites and generated additional diagnostic fragment ions, such as those specific to glycan structures or bracketing O -glycosylation sites, to dissect O -glycosylation patterns. ,, However, HCD does not consistently produce cross-ring cleavage fragments (A and X ions) essential for confirming linkage information between the saccharides . Electron-based dissociation methods, such as electron transfer dissociation and EThcD, ,− can determine the composition of an O -glycan but do not delineate intersaccharide linkages. , Using combinations of MS/MS methods bolsters the confidence of O -glycosite identifications, but ambiguity often remains if two or more glycosylation sites occur in proximity and diagnostic fragments bracketing each site are missing . Of added interest is the generation of sets of high-coverage glycosidic fragment ions, including cross-ring cleavages that best depict the glycan structure, especially when there is a choice among isomeric structures.…”

O-Glycopeptide Truncation Strategy for Heterogeneous O-GalNAc Glycoproteomics Characterization

“…In order to enhance the MS detection and database search for analyzing O-glycopeptides, several studies have applied enzyme-assisted strategies to simplify the O-glycopeptides and/or O-glycans. For instance, in some glycoproteomic studies, broad- and nonspecific proteases (such as proteinase K or pronase E) are proposed for broad-specific proteolysis, while trypsin combined with other specific proteases (such as Glu-C or chymotrypsin) are used for multi-enzyme proteolysis. − In this way, some of the multiply O-glycosylated peptides might be simplified into singly O-glycosylated peptides for better site-specific characterization. However, because of the small structure of O-glycans, singly O-glycosylated peptides may not have sufficient hydrophilicity to be captured by hydrophilic-based enrichment columns, compared to multiply O-glycosylated peptides.…”