Efficient Mapping of Sulfated Glycotopes by Negative Ion Mode nanoLC–MS/MS-Based Sulfoglycomic Analysis of Permethylated Glycans

Abstract

We have previously developed the enabling techniques for sulfoglycomics based on mass spectrometry (MS) analysis of permethylated glycans, which preserves the attractive features of more reliable MS/MS sequencing compared with that performed on native glycans, while providing an easy way to separate and hence enrich the sulfated glycans. Unlike LC-MS/MS analysis of native glycans in negative ion mode that has been more widely in use, the characteristics and potential benefits of similar applications based on permethylated sulfated glycans have not been fully investigated. We report here the important features of reverse phase-based nanoLC-MS/MS analysis of permethylated sulfated glycans in negative ion mode and demonstrate that complementary sets of diagnostic fragment ions afforded can allow rapid identification of various fucosylated, sialylated sulfated glycotopes and definitive determination of the location of sulfate in a way difficult to achieve by other means. A parallel acquisition of both higher collision energy and trap-based MS2 coupled with a product dependent 1 is conceivably the most productive sulfoglycomic workflow currently possible and the manually curated fragmentation characteristics presented here will allow future developments in automating data analysis.

Advancing a High Throughput Glycotope-centric Glycomics Workflow Based on NAnoLC-MS2-product Dependent-MS3 ANAlysis of Permethylated Glycans*

“…We have previously shown that by converting all hydroxyl groups including the carboxylic groups of sialic acids into O-methyl, permethylation allows simple enrichment and identification of a wealth of sulfated glycans occurring at low abundance that would otherwise not be detected (7,8). We further demonstrated that negative ion mode nanoLC-MS/MS can be productively applied in acidic buffer commonly used in proteomics without compromising much the stability and detection sensitivity (9). Low mass MS 2 diagnostic ions for locating the sulfate onto the common Gal␤1-3/4GlcNAc unit, with and without additional sialylation, were identified and we suggested that ambiguity in assignment can conceivably be addressed by additional MS 3 mode that will not suffer from low mass cutoff the way an ion trap based fragmentation would (9).…”

“…We further demonstrated that negative ion mode nanoLC-MS/MS can be productively applied in acidic buffer commonly used in proteomics without compromising much the stability and detection sensitivity (9). Low mass MS 2 diagnostic ions for locating the sulfate onto the common Gal␤1-3/4GlcNAc unit, with and without additional sialylation, were identified and we suggested that ambiguity in assignment can conceivably be addressed by additional MS 3 mode that will not suffer from low mass cutoff the way an ion trap based fragmentation would (9).…”

“…The MS system was operated in negative ion mode for analyses of mono-sulfated O-glycans (m/z mass range 700 -2000, charge state 1). The same Orbitrap resolution and AGC target value were applied for MS 1 but a 5 s top speed mode was used instead for parallel HCD/CID MS 2 data dependent acquisition (9). The AGC target value and NCE for CID MS 2 in the ion trap was set at 1 ϫ 10 4 and 40%, respectively.…”

Distinct substrate specificities of human GlcNAc-6-sulfotransferases revealed by mass spectrometry–based sulfoglycomic analysis

“…To account for the observed sulfoglycomic differences contributed by different GlcNAc6STs, the nanoLC-MS/MS spectra for the few major monosulfated O-glycans commonly found in the parental SW480 and all three transfected cell lines were extracted and manually interpreted to delineate their isomeric structures and location of sulfate based on detecting a combination of previously reported diagnostic ions (38,40,41). In the case of m/z 1025, 1386, and 1474, which seemingly defined the most obvious differences in the MS profiles of monosulfated O-glycans made by cells overexpressing the three GlcNAc6STs ( Fig.…”

“…applied to more problematic glycans, such as the sulfated glycans and/or to derive linkage-specific information requiring advanced MS/MS. Over the last few years, we have steadily established a reasonably robust and sensitive sulfoglycomic platform based initially on MALDI-MS/MS and then nanoLC-MS/MS analyses of permethylated sulfated glycans fractionated away from the otherwise dominant nonsulfated components (36,40,41,45,46). These technical advances now enable us to meaningfully dissect the in vivo specificities of various GlcNAc6STs on O-glycans by sulfoglycomic analysis that focused on a few representative base structures, with a good balance of precision and sensitivity.…”