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Comparison of glycopeptide to released glycan abundances and the influence of glycopeptide charge state on N-linked glycosylation of IgG antibodies

ABSTRACT: We report the comparison of mass-spectral-based abundances of tryptic glycopeptides with fluorescence abundances of released labeled glycans, both derived from therapeutic monoclonal antibodies. These proteins were selected for their very high purities, which assures the origin of all observed released glycans. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances over a dynamic range of 500. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high mannose and biantennary complex galactosylated and fucosylated and, in some cases, sialylated N-glycans. Except for Evolucumab, in-source ions derived from the loss of HexNAc or HexNAc and hexose sugars to HexNAc(4)Hex(4)Fuc(1) glycoform are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances across all mAb samples over a dynamic range of 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated closely to examine the effect of charge state on ion abundances. These results confirmed and refined results for the therapeutic proteins, clearly revealing a linear dependence of relative glycopeptide abundance on the mass of the glycan, with higher charge states favoring higher mass glycans. Overall, our findings indicate that the mass-spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described Glycopeptide Abundance Distribution Spectra GADS representations.

INSTRUMENT(S): Orbitrap Fusion Lumos, timsTOF Pro 2

ORGANISM(S): Human

SUBMITTER: Concepcion Remoroza

PROVIDER: MSV000093562 | MassIVE | Mon Dec 04 13:24:00 GMT 2023

SECONDARY ACCESSION(S): PXD047513

REPOSITORIES: MassIVE

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Project description:The most common congenital disorder of glycosylation (CDG) is caused by pathogenic variants in PMM2 (PMM2-CDG) that reduce phosphomannomutase activity and impair N-glycan synthesis. Patients present early in life with multi-system disabilities. Traditional diagnosis relies on measuring carbohydrate-deficient transferrin (CDT) and confirmation by genetic testing. However, tests for CDT can be normal in some patients, or normalize with age. Because patients also show subtle alterations in serum glycan structures, we considered that site-specific glycosylation changes might be diagnostic. We analyzed serum from 35 individuals with PMM2-CDG for discovery and validation of glycoproteomic changes. We carried out mass spectrometry-based discovery studies to profile the glycoproteome in an initial set of well-characterized affected individuals and matched controls. Next, we developed a streamlined method for analyzing additional affected individuals. Finally, targeted mass spectrometry was used to validate selected N-glycopeptides in an independent set of samples in a blinded fashion. Of the 3,342 N-glycopeptides derived from 284 glycoproteins identified in discovery experiments, individuals with PMM2-CDG exhibited a general decrease in complex-type N-glycans and an increase in truncated, mannose-rich and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls. Notably, this glycopeptide was also detected in five affected individuals with normal CDT, including one individual after liver transplant, two individuals with a known genetic variant associated with mild disease and two individuals with well characterized variants, indicating that this glycopeptide is more sensitive than CDT. Finally, this marker was also detected by targeted analysis in two individuals with variants of uncertain significance (VUS) in PMM2 gene. Overall, we believe that C4-derived Man5GlcNAc2 glycopeptide could serve as a novel next-generation biomarker for more accurate diagnosis and tracking progress of patients in therapeutic trials.

Project description:To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over‑expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper‑mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. Our proteome and transcriptome analyses have been performed during stationary‑phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets. Keywords: Molecular markers, antibiotic resistance, glycopeptides, growth-phase

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Comparison of glycopeptide to released glycan abundances and the influence of glycopeptide charge state on N-linked glycosylation of IgG antibodies

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