Optimising HCD fragmentation energy for TMT-labelled intact glycopeptide samples
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Shisheng Sun
PROVIDER: PXD060000 | iProX | Mon Jan 20 00:00:00 GMT 2025
REPOSITORIES: iProX
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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: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:In this study, we conducted a systematic investigation into a glycoproteomic workflow. Firstly, Label-free and TMT labeling strategies were utilized for intact N-glycopeptide quantification. A total of 2,924 unique intact N-glycopeptides can be quantified after being labeled using TMT approach. Compared to label-free method, TMT labeling for its high precision with the CV averaging 8% and multiplex analysis capabilities. Subsequently, we compared the capacity of ZIC-HILIC and MAX enrichment methods, revealing that the ZIC- HILIC method demonstrated superior performance in enriching intact N-glycopeptides, which can identify 3,004 GPSMs from a single injection of patient-derived xenograft (PDX) sample. Furthermore, we systematically evaluated different higher-energy collisional dissociation (HCD) collision energies, determining that the stepped collision energy HCD (sceHCD) settings of 25, 35, and 45 was the optimal for both the identification and quantification of intact N-glycopeptides.
2025-05-07 | PXD054874 | 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.
2018-07-02 | MSV000082567 | MassIVE
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:Identification results of peptide-spectrum matches supporting Glyco-Decipher manuscript (Glyco-Decipher: Glycan database-independent peptide matching enables discovery of new glycans and in-depth characterization of site-specific N-glycosylation). Recently, several elegant bioinformatics tools have been developed to identify glycopeptides from tandem mass spectra for site-specific glycoproteomics studies. These glycan database-dependent tools have substantially improved glycoproteomics analysis but fail to identify glycopeptides with unexpected glycans. We present a platform called Glyco-Decipher to interpret the glycoproteomics data of N-linked glycopeptides. It adopts a glycan database-independent peptide matching scheme that allows the unbiased profiling of glycans and the discovery of new glycans linked with modifications. Reanalysis of several large-scale datasets showed that Glyco-Decipher outperformed the open search method in glycan blind searching and the popular glycan database-dependent software tools in glycopeptide identification. Our glycan database-independent search also revealed that modified glycans are responsible for a large fraction of unassigned glycopeptide spectra in shotgun glycoproteomics.
2022-03-04 | PXD031025 | 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: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
Project description:Reanalysis of submissions PXD005411, PXD005413, PXD005412, PXD005553, PXD005555, PXD005565 and PXD019937 using Glyco-Decipher. Identification results of peptide-spectrum matches supporting Glyco-Decipher manuscript (Glyco-Decipher: glycan database-independent peptide matching enables discovery of new glycans and in-depth characterization of site-specific N-glycosylation). Recently, several elegant bioinformatics tools have been developed to identify glycopeptides from tandem mass spectra for site-specific glycoproteomics studies. These glycan database-dependent tools have substantially improved glycoproteomics analysis but fail to identify glycopeptides with unexpected glycans. We present a platform called Glyco-Decipher to interpret the glycoproteomics data of N-linked glycopeptides. It adopts a glycan database-independent peptide matching scheme that allows the unbiased profiling of glycans and the discovery of new glycans linked with modifications. Reanalysis of several large-scale datasets showed that Glyco-Decipher outperformed the open search method in glycan blind searching and the popular glycan database-dependent software tools in glycopeptide identification. Our glycan database-independent search also revealed that modified glycans are responsible for a large fraction of unassigned glycopeptide spectra in shotgun glycoproteomics.
2022-03-04 | PXD031032 | Pride
Project description:We investigated how the structural features of N-glycopeptides and the choice of the search engine influence the optimal collision energy, delivering highest identification confidence. We carried out LC-MS/MS measurements using a series of collision energies on a large set of N-glycopeptides with both the glycan and peptide part varied, and studied the behavior of Byonic, pGlyco, and GlycoQuest scores.
2023-10-14 | MSV000093110 | MassIVE