20200520 Khoo KH paper photo 2

Negative Ion Mode nanoLC-ESI-MS/MS Analyses of Permethylated Sulfated Glycans

Negative Ion Mode nanoLC-ESI-MS/MS Analyses of Permethylated Sulfated Glycans

BIO-PROTOCOL. 2020 May 20; 10(10):e3618.
doi: 10.21769/BioProtoc.3618


Yu, Shin-Yi; Cheng, Chu-Wen; Khoo, Kay-Hooi


We have developed enabling techniques for sulfoglycomics based on MALDI-MS mapping and MS/MS sequencing of permethylated sulfated glycans. We then extended further the analytical workflow to C18 reverse phase (RP)-nanoLC-nanoESI-MS/MS analyses of permethylated sulfated glycans in the negative ion mode. The advantages are that extra sulfates on permethylated di- and multiply sulfated glycans will survive in nanoESl conditions to allow detection of multiply charged intact molecular ions, and more comprehensive MS/MS can be performed in an automated fashion at higher sensitivity, compared with MALDI-MS/MS. Parallel higher energy collision dissociation (HCD) and ion trap collision induced dissociation (CID)-based MS2, coupled with product-dependent MS3 in data dependent acquisition mode proved to be highly productive when applied to resolve and identify the isomeric sulfated glycan structures. In-house glycomic data mining software, GlyPick, was developed and used to automate the downstream process of identification and relative quantification of target sulfated glycotopes based on summed intensity of their diagnostic MS2 ions extracted from thousands of HCD-MS2 and/or CID-MS2 data.