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 MS(2) coupled with a product dependent MS(3) 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.

Figure 1. Extracted ion chromatograms of representative permethylated, sialylated O-glycans (A) and N-glycans (B) differing in degree of sulfation

The normalized extracted ion chromatograms from 2 separate LC-MS/MS runs for the mono-sulfated (lower trace) and disulfated or monosulfated but with one under-methylated carboxylic group (upper trace) were aligned and partially superimposed for comparison. In the case of N-glycans, the monosulfated fraction also contained non-sulfated, under-methylated species.

Figure 2. Characteristic low mass HCD fragment ions for determination of the location of sulfate on terminal glycotopes

Useful and diagnostic ions are marked with *. Several ions can be assigned as the expected glycosidic and ring cleavage A ions and denoted according to the established nomenclature²⁹ while additional type of cleavages have previously been observed in MALDI-based high energy cleavages and negative ion mode cleavages^,28, e.g. the E and D ions, for the permethylated glycans. A few others including m/z 181, 195, 234 and 264 are highly reproducible and diagnostic but their origins are uncertain, with probable structures proposed as drawn without implying any mechanistic basis for their formation. The standards were synthesized with linker as drawn here but the respective parent ions and reducing end fragment ions are not considered for current purposes. All gave an abundant ion at m/z 97 corresponding to HSO₄⁻ (not shown) but only those sialylated ones additionally produced a high intensity ion at m/z 111, possibly resulting from the sulfate acquiring a methyl group from the carboxylic group.

Figure 3. Exemplary HCD/CID MS² of permethylated, monosulfated O-glycans (A–B) and CID MS³ of monosulfated terminal glycotopes (C–D)

Each of the existing isomeric structures for the parent O-glycans as can be deduced from their respective HCD and CID MS² spectra, are shown here as structures I–III or IV with associated key fragmentation patterns. Schematic drawings are also provided for the origins of satellite ions associated with the B ions for sulfated lacNAc (m/z 528) and sulfated fucosylated lacNAc (m/z 702) extending from the 6-arm of reducing end GalNAcitol. Loss of 161 u from the parent is consistent with cleavage between C2-C3 of the reducing end GalNAcitol concomitant with elimination of the OMe on the C4. CID MS³ of the sulfated fucosylated lacNAc in (C) clearly confirms location of sulfate on the terminal Hex, but does not rule out sulfate on internal HexNAc. Further loss of 74 u can be assigned as losing the C5-C6 substituents analogous to an ^O,A cleavage and can only be produced if the C6 is not sulfated.

Figure 4. Representative CID MS² spectra of doubly charged permethylated disulfated O-glycans

The corresponding HCD MS² afforded very similar sets of low mass ions not observed in CID. Only the mass region of HCD MS² useful for structural assignments are additionally shown here (D, F). Loss of 98 and 112 u corresponds to loss of sulfate and −48 u corresponds to further concerted loss of the OMe from C3 of the HexNAc, as described in the text.