MALDI-TOF-TOF Analysis of N-Glycans

This analysis of H11 was conducted using an established protocol (31 (link)). N-glycans were released from glycopeptides by PNGase F. PNGase F-resistant N-glycans were subjected to PNGase A digestion. Following porous graphitic carbon (PGC) purification (32 (link)), native glycans were permethylated by adding 100 μL of DMSO-NaOH slurry. Subsequently, the glycan sample was mixed with freshly-prepared 2,5-dihydroxybenzoic acid (DHB) before crystallisation at room temperature. N-glycome spectra were obtained in the positive ionisation mode using an AB 5800 MALDI-TOF-TOF instrument (SCIEX, Concord, Canada). The data were processed using Data Explorer 4.0 (SCIEX) and GlycoWorkbench (v.2.1).

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Wang C., Liu L., Wang T., Liu X., Peng W., Srivastav R.K., Zhu X.Q., Gupta N., Gasser R.B, & Hu M. (2022). H11-induced immunoprotection is predominantly linked to N-glycan moieties during Haemonchus contortus infection. Frontiers in Immunology, 13, 1034820.

Publication 2022

Data Explorer 4.0

Carbon Crystallisation Digestion Dihydroxybenzoic acid Dmso Glycan Glycopeptides Graphitic Maldi Pngase f

Corresponding Organization : University of Melbourne

Other organizations : Huazhong University of Science and Technology, Shanghai Jiao Tong University, Birla Institute of Technology and Science, Pilani, Shanxi Agricultural University, Humboldt-Universität zu Berlin

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Variable analysis

independent variables

PNGase F digestion to release N-glycans from glycopeptides
PNGase A digestion of PNGase F-resistant N-glycans
Porous graphitic carbon (PGC) purification of native glycans
Permethylation of glycans by adding DMSO-NaOH slurry

dependent variables

N-glycome spectra obtained in positive ionisation mode using MALDI-TOF-TOF instrument

control variables

Positive and negative controls not explicitly mentioned

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