LC-MS(n) analysis of isomeric chondroitin sulfate oligosaccharides using a chemical derivatization strategy

Abstract

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS(n). The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS(n) fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS(n) experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS(n) methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.

Figure 1

Derivatized product of [ΔGlcA-GalNAc(6S)-GlcA-GalNAc(6S)-GlcA-GalNAc(6S)-ol] (ΔC6;6;6S-ol), where the symbol Δ indicates 4,5-unsaturation on GlcA at the nonreducing end, the “S” refers to sulfation group, and the “-ol” indicates GalNAc at the reducing end is reduced. The structure shows that there is one acetyl group on the 6 position for each GalNAc indicating the original sulfation sites, while other positions are all methylated

Figure 2

Fragmentation path for derivatized CS trisulfated hexasacharides (a) and MSⁿ spectra for derivatized oligosaccharide ΔC6;6;6S-ol: (b) MS² of [M+2Na]²⁺ (m/z 774.8) and (c) MS³ of [Y₄+Na]⁺ (m/z 1053.4). Symbols: ΔGlcA, GlcA, GalNAc. All product ions labeled on the spectra are singly charged and monosodiated unless otherwise annotated

Figure 3

MSⁿ spectra for two derivatized hexasaccharide standards ΔC4;4;4S-ol (a), (b), (c) and ΔC6;6;6S-ol (d), (e), (f). From top row to bottom row, they are (a), (d) MS³ of [B₂+Na]⁺ (m/z 496.2), (b), (e) MS⁴ of [B₄Y₄+Na]⁺ (m/z 514.2) and (c), (f) MS⁴ of [Y₂+Na]⁺ (m/z 562.2) spectra, representing the three disaccharide units respectively at the nonreducing end, in the middle and at the reducing end. The peak marked with an asterisk represents the diagnostic product ions used for differentiation between 4-sulfated and 6-sulfated GalNAc

Figure 4

Two different MS³ of [B₂+Na]⁺ (m/z 528.2) spectra extracted from the LC-MSⁿ analysis of mixture of derivatized trisulfated hexasaccharides with a saturated nonreducing end. While the product ion spectra are notably different from the hexasaccharide standards with an unsaturated non-reducing end (Figure 3a/d), the same diagnostic ions were observed at m/z 196.2 and 278.2 representing the 4-sulfated GalNAc (a) and the 6-sulfated (b) GalNAc. The peak marked with an asterisk represents the diagnostic product ions used for differentiation between 4-sulfated and 6-sulfated GalNAc

Figure 5

LC-MSⁿ analysis for a derivatized mixture of CS trisulfated hexasaccharides with saturated nonreducing ends. Base peak chromatograms were shown for MS/MS spectra of three disaccharide units: (a) MS³ of [B₂+Na]⁺, (b) MS⁴ of [B₄Y₄+Na]⁺ and (c) MS⁴ of [Y₂+Na]⁺ (black). Extracted single ion chromatograms (SIC) with m/z of diagnostic ions indicate the appropriate GalNAc is 6-sulfated (in red) or 4-sulfated (in purple). The three sulfation sites for each BPC peak can be assigned separately by the color of the three SIC that cover the corresponding peak area