Negative Electron Transfer Dissociation Sequencing of 3-O-Sulfation-Containing Heparan Sulfate Oligosaccharides

Abstract

Among dissociation methods, negative electron transfer dissociation (NETD) has been proven the most useful for glycosaminoglycan (GAG) sequencing because it produces informative fragmentation, a low degree of sulfate losses, high sensitivity, and translatability to multiple instrument types. The challenge, however, is to distinguish positional sulfation. In particular, NETD has been reported to fail to differentiate 4-O- versus 6-O-sulfation in chondroitin sulfate decasaccharide. This raised the concern of whether NETD is able to differentiate the rare 3-O-sulfation from predominant 6-O-sulfation in heparan sulfate (HS) oligosaccharides. Here, we report that NETD generates highly informative spectra that differentiate sites of O-sulfation on glucosamine residues, enabling structural characterizations of synthetic HS isomers containing 3-O-sulfation. Further, lyase-resistant 3-O-sulfated tetrasaccharides from natural sources were successfully sequenced. Notably, for all of the oligosaccharides in this study, the successful sequencing is based on NETD tandem mass spectra of commonly observed deprotonated precursor ions without derivatization or metal cation adduction, simplifying the experimental workflow and data interpretation. These results demonstrate the potential of NETD as a sensitive analytical tool for detailed, high-throughput structural analysis of highly sulfated GAGs. Graphical Abstract.

Keywords: Fourier transform ion cyclotron resonance mass spectrometry; Glycomics; Glycosaminoglycan; Heparan sulfate; Negative electron transfer dissociation; Sulfation.

Figure 1

Size-exclusion chromatography of lyase II digest of H3: a, total ion chromatogram of lyase II digest of H3; b, extracted ion chromatogram (EIC) of m/z 544.99, [M − 2H]²⁻ of lyase II-generated T4; c, EIC of m/z 581.10, [M − H]⁻ of ΔHexA-GlcNS6S-R, R = (CH₂)₅NH₂

Figure 2

NETD cleavage maps and tandem mass spectra of the [M − 4H]⁴⁻ precursor of synthetic HS tetrasulfated tetramers: a, T1, GlcA-GlcNS-IdoA-GlcNS3S6S; b, T2, GlcA-GlcNS6S-IdoA-GlcNS3S.

Figure 3

NETD cleavage maps and tandem mass spectra of the [M − 4H]⁴⁻ precursor of synthetic HS pentasulfated tetramers: a, T3, GlcA-GlcNS-IdoA2S-GlcNS3S6S; b, T4, GlcA-GlcNS6S-IdoA-GlcNS3S6S.

Figure 4

NETD cleavage maps of the [M − 5H]⁵⁻ precursor of synthetic HS hexamers: a, H1, GlcA-GlcNS6S-IdoA-GlcNS3S-GlcA-GlcNS6S; b, H2, GlcA-GlcNS6S-IdoA-GlcNS6S-GlcA-GlcNS6S; c, H3, GlcA-GlcNS6S-IdoA-GlcNS3S6S-GlcA-GlcNS6S.

Figure 5

Size-exclusion chromatography of the resistant tetrasaccharides from HSPIM lyase II digest. a, EICs; b, UV absorbance at 232 nm.

Figure 6

NETD cleavage maps of the lyase resistant tetramers from HSPIM: a, ΔHexA-GlcNAc6S-GlcA-GlcNS3S, [M − 3H]³⁻ ; b, ΔHexA-GlcNAc6S-GlcA-GlcNS3S6S, [M − 4H]⁴⁻ ; c, ΔHexA2S-GlcNAc6S-GlcA-GlcNS3S6S, [M − 4H]⁴⁻ ; d, ΔHexA2S-GlcNS6S-GlcA-GlcNS3S6S, [M − 4H]⁴⁻.