Advanced Assesement Through Intact Glycopeptide Analysis of Infliximab Biologics and Biosimilar
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Eclipse
ORGANISM(S): Infliximab
SUBMITTER: 
Heeyoun Hwang
PROVIDER: MSV000090073 | MassIVE | Wed Aug 03 05:35:00 BST 2022
SECONDARY ACCESSION(S): PXD035788
REPOSITORIES: MassIVE
Similar Datasets
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
2007-03-09 | GSE5188 | GEO
Project description:A comprehensive screening of site-specific N-glycopeptides in serum haptoglobin (Hp), a reporter molecule for aberrant glycosylation in HCC, has been performed to characterize glycopeptide markers for NASH-related HCCs.
2020-05-22 | PXD018502 | Pride
Project description:Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes another protease or O-glycopeptidase for targeted cleavage of tryptic and enriched O-glycopeptides, which increases the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with dense O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. And the enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for MS detection and database search in general bottom-up glycoproteomics. We exploit different enzymatic schemes in the O-glycopeptide truncation strategy for large-scale O-glycoproteome analysis. Ultimately, we comprehensively identify nearly 2000 O-glycopeptides from 391 glycoproteins in a total of 75 µL human serum. Moreover, the truncated O-glycopeptides contribute to site-specific O-glycosylation characterization in stepped high-energy collisional dissociation fragmented LC-MS/MS. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.
2023-05-13 | PXD041041 | JPOST Repository
Project description:Mass spectrometry is the premier tool for identifying and quantifying site-specific protein glycosylation globally. Analysis of intact glycopeptides often requires an enrichment step, after which the samples remain highly complex and exhibit a broad dynamic range of abundance. Here, we evaluated the analytical benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to nano-liquid chromatography mass spectrometry (nLC-MS) for analyses of intact glycopeptide devoid of any enrichment step. We compared the effects of compensation voltage on the transmission of N- and O-glycopeptides derived from heterogeneous protein mixtures using two FAIMS devices. We comprehensively demonstrate the performance characteristics of the FAIMS device for glycopeptide analysis and recommend optimal electrode temperature and compensation voltage (CV) settings for N- and O-glycopeptide analysis. Under optimal CV settings, FAIMS-assisted gas-phase fractionation in conjunction with chromatographic reverse phase separation resulted in a 31% increase in the detection of both N- and O-glycopeptide compared to control experiments without FAIMS. Overall, our results demonstrate that FAIMS provides an alternative means to access glycopeptides without any enrichment providing an unbiased global glycoproteome landscape. In addition, our work provides the framework to verify ‘difficult-to-identify’ glycopeptide features.
2023-10-24 | PXD038673 | Pride
Project description:We present the adaptability of Mascot search engine for automated identification of intact glycopeptide mass spectra. The steps involved in adopting Mascot for intact glycopeptide analysis include: i) assigning unique one letter codes for monosaccharides, ii) linearizing glycan sequences and iii) preparing custom glycoprotein databases. Stepped normalized collision energy (NCE) for HCD mostly provided both the peptide and glycan information in a single MS2 spectrum. Using standard glycoproteins, we showed that Mascot can be adopted for automated annotation of both N- and O-linked glycopeptides. In a large scale validation study, a total of 257 glycoproteins containing 970 unique glycosylation sites and 3447 non-redundant N-linked glycopeptide variants were identified in serum samples. This represent a single tool that collectively allows the i) elucidation of N- and O-linked glycopeptide spectra, ii) matching glycopeptides to known protein sequences, and iii) high-throughput, batch wise analysis of large scale glycoproteomics data sets.
2018-02-08 | PXD005931 | Pride
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.
2024-03-12 | PXD042446 | Pride
Project description:We used transcriptome profiling by RNAseq to identify the gene expression signatures elucidated in S. coelicolor in response to the three different glycopeptide compounds that share high degree of structural similarities and the same primary mode of action: dalbavancin, vancomycin and chlorobiphenyl-vancomycin.
2021-02-15 | E-MTAB-9846 | biostudies-arrayexpress
Project description:Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterisation of site-specific glycosylation of proteins remains analytically challenging. Collision induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but loss of labile modifications render CID inappropriate for detailed characterisation of site-specific glycosylation. Electron-based dissociation (ExD) methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localisation, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O- and N-glycopeptides.
2024-07-04 | PXD050173 | Pride
Project description:Recent advances in software-driven glycopeptide identification in LC-MS/MS-based N-glycoproteomics have facilitated biochemical studies reporting thousands of intact N-glycopeptides, i.e. N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, we explore the efficiency of site-specific glycoprofiling using the PTM-centric search-engine Byonic relative to manual expert annotation. To allow an appropriately deep comparison, the study utilised typical glycoproteomics acquisition and data analysis strategies, but of a single glycoprotein, the uncharacterised N-glycosylated (Asn160, Asn268 and Asn302) human basigin. Detailed site-specific reference glycoprofiles of purified basigin were manually established using ion trap CID-MS/MS and high-resolution Q-Exactive Orbitrap HCD-MS/MS acquisition of tryptic N-glycopeptides and released N-glycans. The basigin N-glycosylation sites, which showed extensive micro- and macro-heterogeneity, were then glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS data. The glycoprofiling efficiencies were assessed against the site-specific reference glycoprofiles and target and decoy proteome databases. The search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided very high glycoprofiling accuracy and coverage (both >80%) and low peptide FDRs (<1%). The data complexity, search parameters including search space (proteome/glycome size), mass tolerance and peptide modifications, and confidence thresholds affected the glycoprofiling efficiency and analysis time. Automated identification of peptide modifications (methionine oxidation/carbamidomethylation) that coincide with monosaccharide mass differences (Fuc/Hex/HexNAc) and accurately distinguishing isobaric (Hex1NeuAc1-R/Fuc1NeuGc1-R) or near-isobaric (NeuAc1-R/Fuc2-R) monosaccharide sub-compositions remain challenging, arguing particular attention to such “difficult-to-identify” N-glycopeptides. The presented analysis provides valuable insights into automated glycopeptide identification; knowledge that facilitates further developments in FDR-based glycoproteomics.
2016-08-17 | PXD004243 | Pride
Project description:We developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min. It showed higher enrichment specificity, more intact glycopeptide identifications, and better quantitative reproducibility than the traditional manual method using micro-tip enrichment devices. We further applied this method to investigate the serum site-specific N-glycosylation changes between four patients with pancreatic cancer and seven healthy controls. The principal component analysis of intact N-glycopeptides showed a good clustering across cancer and normal groups. And we found that the site-specific glycoforms, mono-fucosylated and non-sialylated oligosaccharides, on IgG1 site 180 expressed a significant decrease in pancreatic cancer patients compared to healthy controls. Together, the automated method is a powerful tool for site-specific N-glycoproteomics analysis of complex biological samples, and it has great potential for clinical utilities.
2021-04-06 | PXD025151 | JPOST Repository