Electrospray ionization in‐source decay of <i>N</i>‐glycans and the effects on <i>N</i>‐glycan structural identification

Liew, Chia Yen; Chen, Jien-Lian; Ni, Chi-Kung

doi:10.1002/rcm.9352

Cited by 11 publications

(9 citation statements)

References 54 publications

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“…These N -glycans were then sent into two-dimensional HPLC for further purification and isomer separation. A study demonstrated that approximately 1% of N -glycans undergo in-source decay and crack into small N -glycans during the ionization process of electrospray ionization . If a high concentration of large N -glycans is mixed with a low concentration of small N -glycans in the ESI solution, ESI in-source decay of large N -glycans may interfere with the structural identification of small N -glycans.…”

Section: Resultsmentioning

confidence: 99%

Chromatograms and Mass Spectra of High-Mannose and Paucimannose N-Glycans for Rapid Isomeric Identifications

Liew,

Chen,

Lin

et al. 2024

J. Proteome Res.

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N-Linked glycosylation is one of the most essential post-translational modifications of proteins. However, N-glycan structural determination remains challenging because of the small differences in structures between isomers. In this study, we constructed a database containing collision-induced dissociation MS n mass spectra and chromatograms of high-performance liquid chromatography for the rapid identification of high-mannose and paucimannose N-glycan isomers. These N-glycans include isomers by breaking of arbitrary numbers of glycosidic bonds at arbitrary positions of canonical Man 9 GlcNAc 2 N-glycans. In addition, some GlcMan n GlcNAc 2 N-glycan isomers were included in the database. This database is particularly useful for the identification of the N-glycans not in conventional Nglycan standards. This study demonstrated the application of the database to structural assignment for high-mannose N-glycans extracted from bovine whey proteins, soybean proteins, human mammary epithelial cells, and human breast carcinoma cells. We found many N-glycans that are not expected to be generated by conventional biosynthetic pathways of multicellular eukaryotes.

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Section: Resultsmentioning

confidence: 99%

Chromatograms and Mass Spectra of High-Mannose and Paucimannose N-Glycans for Rapid Isomeric Identifications

Liew,

Chen,

Lin

et al. 2024

J. Proteome Res.

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“…Next, the N -glycans were separated according to their sizes by HPLC using a TSKgel Amide-80 column; the chromatogram is shown in Figure (a). The separation of N -glycans by different sizes avoids the interference in structural determination of small N -glycans by large N -glycans due to the ESI in-source decay . For example, Figure (a) shows that the retention time of 19–23 min mainly consists of Man 6 GlcNAc 2 ( m / z 1419) and a small amount of Man 5 GlcNAc 2 ( m / z 1257).…”

Section: Resultsmentioning

confidence: 99%

Identification of the High Mannose N-Glycan Isomers Undescribed by Conventional Multicellular Eukaryotic Biosynthetic Pathways

et al. 2023

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N-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote N-glycan biosynthesis suggests high mannose N-glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. According to conventional biosynthetic pathways, four Man7GlcNAc2 isomers, three Man6GlcNAc2 isomers, and one Man5GlcNAc2 isomer are generated during this process. In this study, we applied our latest mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MS n ), to re-examine high mannose N-glycans extracted from various multicellular eukaryotes which are not glycosylation mutants. LODES/MS n identified many high mannose N-glycan isomers previously unreported in plantae, animalia, cancer cells, and fungi. A database consisting of retention time and CID MS n mass spectra was constructed for all possible Man n GlcNAc2 (n = 5, 6, 7) isomers that include the isomers by removing arbitrary numbers and positions of mannose from canonical N-glycan, Man9GlcNAc2. Many N-glycans in this database are not found in current N-glycan mass spectrum libraries. The database is useful for rapid high mannose N-glycan isomeric identification.

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“…The CID sequences in LODES were derived from carbohydrate dissociation mechanisms [42][43][44][45] . LODES enables us to determine the D, E, F, and G, according to the dissociation mechanisms of hexose [27][28][29][30][31][41][42][43] .…”

Section: A Databases Of Man N Glcnac 2 (N=5 6 7) Isomersmentioning

confidence: 99%

“…The amide-80 column separated N-glycans into different sizes, as illustrated in Fig. 2(a), to eliminate the interference by ESI in-source decay 41 . The fractions collected from the eluents of amide-80 column were sent into the second HPLC using a PGC Hypercarb column (2.1 mm × 100 mm, particle size of 3 µm, Thermo Fisher Scientific, Waltham, MA, USA) for N-glycan isomer separation.…”

Section: (D) Separation Of N-glycan Isomersmentioning

confidence: 99%

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Identification of novel high mannose N-glycan isomers undescribed by current multicellular eukaryotic biosynthetic pathways

Liew

Luo

et al. 2022

Preprint

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N-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote N-glycan biosynthesis suggests high mannose N-glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. As a part of post-translational modifications, lipid dolichol-phosphate linked oligosaccharide Glc3Man9GlcNAc2 is transferred to proteins, and glucoses and mannose are sequentially removed by various ER- and Golgi-localized glucosidases and -1,2-mannosidases. According to reported biosynthetic pathways, four Man7GlcNAc2 isomers, three Man6GlcNAc2 isomers, and one Man5GlcNAc2 isomer are generated during this process. In this study, we applied our latest mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn) to re-examine high mannose N-glycans extracted from various multicellular eukaryotes. LODES/MSn identified many high mannose N-glycan isomers previously unreported in plantae, animalia, cancer cells, and fungi. Importantly, their synthesis is yet described by known biosynthetic pathways, thereby suggesting additional and unidentified pathways for these N-glycans isomers in multicellular eukaryotic cells.

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Electrospray ionization in‐source decay of N‐glycans and the effects on N‐glycan structural identification

Cited by 11 publications

References 54 publications

Chromatograms and Mass Spectra of High-Mannose and Paucimannose N-Glycans for Rapid Isomeric Identifications

Chromatograms and Mass Spectra of High-Mannose and Paucimannose N-Glycans for Rapid Isomeric Identifications

Identification of the High Mannose N-Glycan Isomers Undescribed by Conventional Multicellular Eukaryotic Biosynthetic Pathways

Identification of novel high mannose N-glycan isomers undescribed by current multicellular eukaryotic biosynthetic pathways

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