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Abstract
Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans including microheterogeneity at the same amino acid residue (glycosite). Top-down mass spectrometry (MS) allows precise identification and localization of glycans on intact proteins, and coupling top-down MS with chromatography allows time-resolved characterization of glycoforms. Here, we couple ultraviolet photo-dissociation (UVPD) to hydrophilic interaction chromatography (HILIC) to advance the characterization of glycoproteins ranging from 15-34 kDa, offering site localization of glycans, providing sequence coverages up to 93% and relative quantitation of individual glycoforms.
Supplementary materials
Title
PDF SI
Description
Protein sequences, masses, and catalog numbers, LC gradients, comparison of glycoform abundances to literature values, PLRP vs HILIC separations for RNase B, deconvoluted RNase B MS1 spectra, comparison of chromatographic peak shape for RNase B MS1 only and UVPD runs, ESI mass spectra of HA collected without LC separation, sequence coverage maps and extracted ion chromatograms for RNase B (HCD, ETD, EThcD, and UVPD), HA (UVPD only) and RBDs (UVPD only).
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Title
Spreadsheet SI
Description
Identified fragment ions in MS2 spectra for RNase B (HCD, ETD, EThcD, and UVPD), HA (UVPD only) and RBDs (UVPD only). Relative quantitation results for RNase B, HA, and the RBDs.
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