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 nanoESI 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.

Sulfated glycans are barely detectable in routine mass spectrometry (MS)-based glycomic analysis due to ion suppression by the significantly more abundant neutral glycans in the positive ion mode, and sialylated non-sulfated glycans in the negative ion mode, respectively. Nevertheless, the negative charge imparted by sulfate can be advantageous for selective detection in the negative ion mode if the sialic acids can first be neutralized. This is most conveniently achieved by a concerted sample preparation workflow in which permethylation is followed by solid phase fractionation to isolate the sulfated glycans prior to MS analysis. Importantly, we demonstrated that conventional NaOH/DMSO slurry permethylation method can retain the sulfates. Instead of extracting permethylated glycans into chloroform for sample clean-up, reverse phase C18 cartridge coupled with self-packed amine-tip or mixed mode weak anion exchange cartridge can be utilized to obtain in good yield the non-sulfated, mono-sulfated, and multiply sulfated permethylated glycans in separate fractions for sulfoglycomic analysis.