Simply extending the EThcD MS/MS range increases the confidence in N-glycopeptide identification.
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Cricetulus Griseus (ncbitaxon:10029) Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Albert J.R. Heck
PROVIDER: MSV000083710 | MassIVE | Mon Apr 22 06:41:00 BST 2019
REPOSITORIES: MassIVE
Similar Datasets
Project description:Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Furthermore, N-glycoproteomics has made significant progress in the last few years, and there is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and comment on their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.
2020-07-20 | PXD017646 | Pride
Project description:Site-specific N-glycosylation characterization requires intact N-glycopeptide analysis based on suitable tandem mass spectrometry (MS/MS) method. Electron-transfer/higher-energy collisional dissociation (EThcD), stepped collision energy/higher-energy collisional dissociation (sceHCD), and higher-energy collision dissociation-product-dependent electron-transfer dissociation (HCD-pd-ETD) have emerged as valuable approaches for clinical glycoproteomics. However, each of them incurs some compromise, necessitating the performance comparisons when applied to the analysis of complex clinical samples (e.g., plasma, urine, cells, and tissue). Herein, we developed a hybrid mass spectrometry fragmentation method, EThcD-sceHCD, by combining EThcD and sceHCD. Moreover, we compared the performance of this method with those previous approaches (EThcD, sceHCD, HCD-pd-ETD, and sceHCD-pd-ETD) in the intact N-glycopeptide analysis, and determined its applicability for clinical N-glycoproteomic study.
2022-04-04 | PXD030288 | Pride
Project description:Dissecting site-specific functions of O-glycosylation requires simultaneous identification and quantification of differentially ex-pressed O-glycopeptides by mass spectrometry. However, different dissociation methods have not been systematically compared in their performance in terms of identification, glycosite localization and quantification with isobaric labeling. Here, we conducted this comparison with higher-energy collision dissociation (HCD), electron-transfer/collision-induced dissociation (ETciD) and electron transfer/higher-energy collisional dissociation (EThcD), concluding that ETciD with optimal supplemental activation re-sulted in most identifications and unambiguous site localizations. We later described a pseudo EThcD strategy that in silico com-bines ETciD spectrum with HCD spectrum acquired sequentially for the same precursor, which combines the identification ad-vantage of ETciD with the superior reporter ion quality of HCD. We demonstrated its improvements in identifications and quantification of isobaric mass tag-labeled O-glycopeptides and showcased the discovery of the specific glycosylation sites of GalNAc tansferase 11 (GalNAc-T11) in HepG2 cells.
2021-11-25 | PXD025407 | Pride
Project description:N-glycoproteins are involved in various biological processes. More than one-third of plasma tumor protein biomarkers approved by the Food and Drug Administration (FDA) are glycoproteins which would enhance the specificity and/or sensitivity of cancer diagnosis. Therefore, the characterization of plasma N-glycoproteome is essential. In previous studies, we developed an integrated method based on combinatorial peptide ligands library (CPLL) and stepped collision energy/higher-energy collisional dissociation (sceHCD) for in-depth and comprehensive N‑glycoproteome profiling of human plasma. Recently, we presented a new mass spectrometry fragmentation method (EThcD-sceHCD) which outperformed sceHCD in the accuracy of identification. Herein, we integrated CPLL into EThcD-sceHCD, and compared the performance of EThcD-sceHCD with those previous approaches (EThcD and sceHCD) in pooled prostatic cancer (PCa) patients’ plasma intact N-glycopeptide analysis. We found that EThcD-sceHCD outperformed other methods in both the depth and accuracy of intact N-glycopeptides identification, and sceHCD and EThcD-sceHCD have good complementarity. Combining sceHCD and EThcD-sceHCD can cover almost all glycoproteins and intact N-glycopeptides. Our study holds great potential for human plasma biomarker discovery.
2022-08-12 | PXD030622 | Pride
Project description:Use of EThcD, CID, HCD and high energy HCD to identify protein cross-links
2020-06-18 | PXD018310 | Pride
Project description:Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma and monoclonal gammopathy of renal significance. Because of the complicated purification method and the low stoichiometry of purified protein and glycans, site-specific N-glycosylation characterization for monoclonal immunoglobulin is still challenging. Studies reporting the N-glycosylation profiles for this monoclonal immunoglobulin have been rare until now. Therefore, in this study, we presented an integrated workflow for micro serum monoclonal IgA and IgG purification from patients with multiple myeloma in the HYDRASYS system, in-agarose-gel digestion, LC‒MS/MS analysis without intact N-glycopeptide enrichment, and compared the identification performance of different mass spectrometry dissociation methods (EThcD-sceHCD, sceHCD, EThcD and sceHCD-pd-ETD). The results showed that EThcD-sceHCD was a better choice for site-specific N-glycosylation characterization of micro in-agarose-gel immunoglobulin proteins (~2 μg) because it can cover more unique intact N-glycopeptides (37 and 50 intact N-glycopeptides from IgA1 and IgG2, respectively) and provide more high-quality spectra than sceHCD, EThcD and sceHCD-pd-ETD. We demonstrated the benefits of the alternative strategy in site-specific N-glycosylation characterizing micro disease-related proteins obtained from bands separated by electrophoresis. This work could promote the development of clinical N-glycoproteomics and related immunology.
2022-09-13 | PXD035757 | Pride
Project description:N-glycosylation is an essential post-translational modification (PTM) of human milk proteins for the promotion of infant health. N-glycosylation is relevant regarding proteolytic susceptibility and functions as a competitive inhibitor of pathogen binding and immunomodulators. Due to the individual uniqueness of human milk, the overall complexity and temporal changes of N-glycosylation, the analysis for site-specific information of this PTM must take these factors into consideration. Here we demonstrate, by adapting the automated platform and hydrophilic-interaction chromatography (HILIC)-based cartridge with 150 μg of milk protein digestion, that proteome-wide N-glycopeptides can be reproducibly enriched in technical triplicates and biological samples across lactation. With higher energy c-trap dissociation (HCD) triggered electron-transfer/higher-energy collision dissociation (EThcD), we were able to map, to our knowledge, the most comprehensive human milk N-glycoproteome to date. We found 1697 glycopeptides from 110 glycoproteins and 191 glycosites of which 43 novel sites were not yet reported in experimental evidence. We next quantified the glycopeptides in targeted monitoring mode with scheduled selective ion monitoring (SIM) and parallel reaction monitoring (PRM). Co-trending HCD fragment ions enabled us to pinpoint the MS1 extracted ion chromatogram (XIC) of intact glycopeptide. In a 90 min gradient, we could measure 318 glycopeptides from 51 glycoproteins, catching at least 10 data points in their chromatographic peaks. We observed distinctive quantitative site-specific glycosylation trends, indicating the potential of specific temporal changes associated with functions that support the developing infant’s health.
2021-09-08 | PXD013764 | Pride
Project description:Protein N-glycosylation is an essential post-translational modification (PTM) frequently occurring on human milk proteins. These PTMs assist in protecting the infant's health and function amongst others as competitive inhibitors of pathogen binding and immunomodulators. Due to the individual uniqueness of each mother’s milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein- and N-site-specific quantitative information. Here we describe an automated platform using HILIC-based cartridges enabling the proteome-wide monitoring of N-glycopeptides using just 150 μg of breast milk protein digestion. Reproducibility in enrichment and quantitation is demonstrated in triplicate measurements of milk samples taken along an extended lactation period. Using higher energy c-trap dissociation (HCD) triggered electron-transfer higher-energy collision dissociation (EThcD), we were able to map around 1700 glycopeptides from 110 glycoproteins occurring on 191 glycosites, of which 43 sites were not before reported with experimental evidence. We next quantified many of these glycopeptides using a targeted proteomics approach with scheduled selective ion monitoring (SIM) and parallel reaction monitoring (PRM). In a 90 min gradient, we could quantitatively monitor 318 glycopeptides originating from 51 glycoproteins. Although each glycoprotein, each N-glycosylation site and each glycan attached revealed its own dynamic changes, we did observe a few general trends. Overall fucosylation especially terminal fucosylation increased across the lactation period.
2020-11-07 | PXD022415 | panorama
Project description:Antibody–drug conjugates (ADCs) are formed by binding of cytotoxic drugs to monoclonal antibodies (mAbs) through chemical linkers. A comprehensive evaluation of the critical quality attributes (CQAs) of ADCs is vital for drug development but remains challenging owing to ADC structural heterogeneity than mAbs. Drug conjugation sites can considerably affect ADC properties, such as stability and pharmacokinetics, however, few studies have focused on method development in this area owing to technical challenges. Hybrid electron-transfer/higher-energy collision dissociation (EThcD) produces more fragment ions than conventional high collision dissociation (HCD) fragmentation, which aid in identifying and localizing post-translational modifications (PTMs). Herein, we systematically employ EThcD to assess the fragmentation mode impact on conjugation site characterization for randomly conjugated and site-specific ADCs. EThcD generates more fragment ions in tandem mass spectrometry (MS/MS) spectra compared with HCD. Additional ions aid in pinpointing the correct conjugation sites that bear complex linker payload structures. Our study may contribute to the quality control of various preclinical and clinical ADCs.
2023-02-14 | PXD040111 | iProX
Project description:We demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 98% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.
2021-01-11 | PXD023419 | Pride