Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-CoV-2 Spike Receptor Binding Domain

ABSTRACT: Online LCMS intact glycoform separation of SARS-CoV-2 spike receptor binding domain proteins expressed in HEK-293 cells from the vendors Sino Biological or RayBiotech. C2 RPLC, CZE, and HILIC separation methods are compared as well as top-down and bottom-up analysis to define RBD glycosylation patterns. Intact proteins were buffer exchanged into 100 mM ammonium acetate before online LC separation in triplicate. ETD top-down analysis was performed after PNGase F N-glycan removal. Bottom-up analysis was performed using cysteine alkylation with iodoacetamide and trypsin digestion for RayBiotech RBDs and a combination of Glu-C and trypsin digestion for Sino Biological RBDs. Intact data (in triplicate per an RBD) were processed using PMI Intact for all glycoform separation experiments and output in csv format. Glycopeptide data were processed with PMI Byonic in the folder PMI_Byonic_results which can be found inside the directory named "other" as well as the processed intact data.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos, Q Exactive HF, Orbitrap Exploris 480

ORGANISM(S): Sars Coronavirus (ncbitaxon:227859) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Mowei Zhou  

PROVIDER: MSV000088601 | MassIVE | Tue Dec 21 16:44:00 GMT 2021

REPOSITORIES: MassIVE