Solid-phase reductive amination for glycomic analysis

Jiang, Kuan; Zhu, He; Xiao, Cong; Liu, Ding; Edmunds, Garrett; Wen, Liuqing; Ma, Cheng; Li, Jing; Wang, Peng George

doi:10.1016/j.aca.2017.01.049

Cited by 19 publications

(11 citation statements)

References 39 publications

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“…Three different high‐mannose‐type glycoforms were allocated to these N ‐glycopeptides (HexNAc 2 Hex 5,6,8 —also known as Man5, Man6, Man8). Detection of these glycoforms is in good agreement with literature reports, as these are the major ones present one RNase B (Figure S8, Supporting Information). The two lower‐abundant glycoforms, Man7 and Man9, were not detected in the present work.…”

Section: Resultssupporting

confidence: 91%

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The Fine Art of Destruction: A Guide to In‐Depth Glycoproteomic Analyses—Exploiting the Diagnostic Potential of Fragment Ions

et al. 2018

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The unambiguous mass spectrometric identification and characterization of glycopeptides is crucial to elucidate the micro-and macroheterogeneity of glycoproteins. Here, combining lower and stepped collisional energy fragmentation for the in-depth and site-specific analysis of Nand O-glycopeptides is proposed. Using a set of four representative and biopharmaceutically relevant glycoproteins (IgG, fibrinogen, lactotransferrin, and ribonuclease B), the benefits and limitations of the developed workflow are highlighted and a state-of-the-art blueprint for conducting high-quality in-depth Nand O-glycoproteomic analyses is provided. Further, a modified and improved version of cotton hydrophilic interaction liquid chromatography-based solid phase extraction for glycopeptide enrichment is described. For the unambiguous identification of N-glycopeptides, the use of a conserved yet, rarely employed-fragmentation signature [M peptide +H+ 0,2 X GlcNAc] + is proposed. It is shown for the first time that this fragmentation signature can consistently be found across all N-glycopeptides, but not on O-glycopeptides. Moreover, the use of the relative abundance of oxonium ions to retrieve glycan structure information, for example, differentiation of hybrid-and high-mannose-type N-glycans or differentiation between antenna GlcNAc and bisecting GlcNAc, is systematically and comprehensively evaluated. The findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics.

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Section: Resultssupporting

confidence: 91%

“…RNase B is a globular glycoprotein that is solely N ‐glycosylated. It harbors only one N ‐glycosylation site, which is occupied by high‐mannose‐type N ‐glycans . In this study, a total of six tryptic N ‐glycopeptides of RNase B were identified (Figure S2, pp.…”

Section: Resultsmentioning

confidence: 91%

The Fine Art of Destruction: A Guide to In‐Depth Glycoproteomic Analyses—Exploiting the Diagnostic Potential of Fragment Ions

et al. 2018

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“…Oligosaccharide standards with Neu5Ac were synthesized according to our previous reports [30,31]. Oligosaccharide derivatives with Neu5Gc were obtained from Chemily Glycoscience (Atlanta, GA, USA).…”

Section: Methodsmentioning

confidence: 99%

Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS

Jiang

Zhu

et al. 2017

Analytica Chimica Acta

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Protein glycosylation mediates a wide range of cellular processes, affecting development and disease in mammals. Deciphering the “glycocodes” requires rapid, sensitive and in-depth characterization of diverse glycan structures derived from biological samples. In this study, we described a two-step derivatization strategy termed linkage-specific sialic acid permethylation (SSAP) consisting of dimethylamination and permethylation for the improved profiling of glycosylation by matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS). High linkage-specificity (~99%) of SSAP to both the two most common forms of sialic acid, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), permitting direct discrimination of α2,3- and α2,6-linked sialic acids in MALDI-TOF MS. The enhanced intensity (>10-fold) and increased detection limit (>10-fold) of derivatized glycans were valued for sensitive glycomics. Moreover, the good compatibility and reaction efficiency of the two steps of SSAP allowed rapid sample preparation (<2 h), benefiting robust analysis of glycans in a high-throughput manner. The SSAP strategy was further applied to investigate the protein glycosylation of human serum associated with rheumatoid arthritis (RA). It was demonstrated that the relative abundances of individual glycans were different in RA negative and RA positive samples, and meanwhile the RA patient/control ratios of both α2,3- and α2,6-sialylated glycans tended to elevate accompanied with the increase of sialylation. Those findings of the glycosylation changes occurred in human serum protein may contribute to the diagnosis of RA. Herein, SSAP derivatization combined with MALDI-TOF MS exhibits unique advantages for glycomic analysis and shows potential in glycosylation profiling of therapeutic proteins and clinical glycan biomarker discovery.

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“…The accuracy of quantitation may also be affected by impurities in the sample matrices. Alternatively, the released glycans were analyzed using matrix-assisted laser desorption ionization–mass spectroscopy (MALDI-MS) or liquid chromatography–electrospray ionization–mass spectroscopy (LC-ESI-MS) after permethylation or derivatization of the reducing end. − While derivatization such as permethylation could improve the ionization efficiency, they often introduced higher variability during the sample preparation due to the additional chemical steps and incomplete reactions. In addition, the permethylated glycan products yielded poor isomeric separation .…”

mentioning

confidence: 99%

Deep Structural Analysis and Quantitation of O-Linked Glycans on Cell Membrane Reveal High Abundances and Distinct Glycomic Profiles Associated with Cell Type and Stages of Differentiation

Goonatilleke

Wongkham

et al. 2020

Anal. Chem.

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Proteins on cell membrane are modified by N- and O-glycans. N-Glycans have been extensively characterized using advanced separation and mass spectrometry techniques. However, O-glycans remain a challenge, because of the lack of universal enzymes to release them and the large background abundances of N-glycans. Here, we report a method for in-depth structural analysis and quantitation of O-glycans derived from human cell membrane. O-Glycans were chemically released from isolated cell membrane glycoproteins following N-glycan and lipid/glycolipid removal by PNGase F digestion and Folch extraction, respectively. Released O-glycans were purified by an optimized protocol to eliminate interference from small molecules and degraded proteins. Cell surface O-glycans were then analyzed using a nanoLC-chip-QTOF mass spectrometer with a porous graphitized carbon (PGC) column, while the N-glycans and glycolipids isolated from the same cell membrane fractions were analyzed in parallel using previously reported methods. The monosaccharide compositions and linkages of the detected O-glycans were identified by exoglycosidase digestion facilitated with tandem mass spectrometry (MS/MS). Using this method, we identified 44 cell membrane O-glycan isomers with MS/MS, and, among them, we unambiguously characterized 25 O-glycan structures with exoglycosidase digestion to create a library with their complete structures, accurate masses, and retention times. In this process, we identified and characterized unexpected mannose oligomers that are α(1–2/3) linked. This library enabled the identification and quantification of unique cell surface O-glycans from different cell lines and the study of specific O-glycan changes during cell differentiation.

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Solid-phase reductive amination for glycomic analysis

Cited by 19 publications

References 39 publications

The Fine Art of Destruction: A Guide to In‐Depth Glycoproteomic Analyses—Exploiting the Diagnostic Potential of Fragment Ions

The Fine Art of Destruction: A Guide to In‐Depth Glycoproteomic Analyses—Exploiting the Diagnostic Potential of Fragment Ions

Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS

Deep Structural Analysis and Quantitation of O-Linked Glycans on Cell Membrane Reveal High Abundances and Distinct Glycomic Profiles Associated with Cell Type and Stages of Differentiation

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