Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Simply extending the EThcD MS/MS range increases the confidence in N-glycopeptide identification.

ABSTRACT: Glycoproteomics raw files containing all classes of N-glycopeptides. Each file has been run as HCD-pd-EThcD where EThcD was triggered upon observation of signature glycopeptide oxonium ions. Additionally each file was measured with 2 different m/z ranges; standard (up to 2000 m/z for both HCD and EThcD) and extended (up to 4000 m/z for HCD and EThcD). CHOKO_FT_IMAC_SAX denotes SAX enriched glycopeptides from CHO cells containing mainly complex multiply sialylated N-glycopeptides. CHOKO_IMAC contains glycopeptides modifed with mannose-6-phosphate. TestGly refers to human milk glycopeptides that contain complex type N-glycopeptides with multiple fucose residues.

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

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Optimal Dissociation Methods Differ for N- and O-glycopeptides

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

EThcD-sceHCD-MS/MS-based Clinical N-glycoproteomics

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

Comprehensive human plasma N-glycoproteome profiling based on EThcD-sceHCD-MS/MS

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

A systematic evaluation of fragmentation methods for unlabeled and isobaric mass tag-labeled O-glycopeptides

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

Site-specific N-glycosylation Characterization of Micro Disease-related Proteins Based on EThcD-sceHCD-MS/MS

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

Conjugation site characterization of antibody–drug conjugates using electron-transfer/higher-energy collision dissociation (EThcD)

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 |

A study into the ADP-ribosylome of IFN--stimulated THP-1 human macrophage-like cells identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation

Project description:ADP-ribosylation is a post-translational modification that, until recently, has remained elusive to study at the cellular level. Previously dependent on radioactive tracers to identify ADP-ribosylation targets, several advances in mass spectrometric workflows now permit global identification of ADP-ribosylated substrates. In this study, we capitalized on two ADP-ribosylation enrichment strategies, and multiple activation methods performed on the Orbitrap Fusion Lumos, to identify IFN--induced ADP-ribosylation substrates in macrophages. The ADP-ribosyl binding protein, Af1521, was used to enrich ADP-ribosylated peptides, and the anti-poly-ADP-ribosyl antibody, 10H, was used to enrich ADP-ribosylated proteins. ADP-ribosyl-specific mass spectra were further enriched by an ADP-ribose product ion triggered EThcD and HCD activation strategy, in combination with multiple acquisitions that segmented the survey scan into smaller ranges. HCD and EThcD resulted in overlapping and unique ADP-ribosyl peptide identifications, with HCD providing more peptide identifications but EThcD providing more reliable ADP-ribosyl acceptor sites. Our acquisition strategies also resulted in the first ever characterization of ADP-ribosyl on three poly-ADP-ribose polymerases, ARTD9/PARP9, ARTD10/PARP10, and ARTD8/PARP14. IFN- increased the ADP-ribosylation status of ARTD9/PARP9, ARTD8/PARP14, and proteins involved in RNA processes. This study therefore summarizes specific molecular pathways at the intersection of IFN- and ADP-ribosylation signaling pathways.

2020-01-27 | PXD011690 | Pride

Global-scale enrichment and identification of O-GlcNAc modified peptides in Arabidopsis seedling (S1 Part 1: HCD and EThcD part from Eclipse)

Project description:O-GlcNAcylation is a crucial post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. However, our understanding of this PTM in plants lags behind animals. To bridge this knowledge gap, we performed a global scale enrichment of O-GlcNAc modified peptides after LWAC enrichment on Col seedlings. A 15N-labeled spy-4 is also included in the samples for comparison. Enriched samples were subjected to extensive reverse high pH fractionation followed by LC-MS-MS analysis on Thermo Fisher Orbitrap mass spectrometer (QExactive HF) using HCD modes (Files initiate with Q) or on Thermo Fisher Orbitrap mass spectrometer Eclipse with ETD using HCD 204 triggered EtHCD.

2024-05-21 | PXD044701 | Pride

Comparison of Intact Glycopeptide Enrichment Methods Using Strong Anion Exchange and Hydrophilic Interaction Liquid Chromatography

Project description:Heterogeneity of protein glycosylation poses great challenge for analysis that is key to un-puzzle systems glycobiology in diseases. Resolving this conundrum requires global enrichment of glycopeptides for identification and quantitation. To this aim, hydrophilic interaction chromatography (HILIC) has been proved to be an effective approach to enrich N-linked glycopeptides (N-glycopeptides). However, its effectiveness to enrich O-linked glycopeptides (O-glycopeptides) and isobaric labelled glycopeptides remains unclear. Here, we studied three different cartridges in HILIC mode. It was found that removal of N-glycosylation prior to enrichment improved identification of O-glycopeptides. It was noted that increased yield and number of glycosylation identification were seen when using cartridges having materials for strong anion exchange (SAX) chromatography. Remarkably, O-glycopeptides were found to be selectively enriched by HILIC with further improved enrichment being seen when Retain AX cartridge being used. Of particular note, isobaric labelled glycopeptides could be readily enriched by SAX cartridges in HILIC mode to enable quantitative glycoproteomics. It is anticipated that the use of SAX cartridges will facilitate broad applications of qualitative and quantitative glycoproteomics in diseases.

2017-10-19 | PXD005145 | Pride

Identify Yeast Proteasome Cross-links with Multiple Ion Fragmentation Methods

Project description:Use of EThcD, CID, HCD and high energy HCD to identify protein cross-links

2020-06-18 | PXD018310 | Pride