Quantification of isomers from a mixture of twelve heparin and heparan sulfate disaccharides using tandem mass spectrometry

Abstract

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) have been implicated in clinically relevant processes such as hemostasis, infection, development, and cancer progression, through their interactions with proteins. Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MSn) were combined to identify and quantify 12 HSGAG disaccharides that can be generated by enzymatic depolymerization with heparin lyases. This technique includes free amine-containing disaccharides that had previously been observed in MSn but not quantified. Our methods use diagnostic product ions from MSn spectra of up to three isomeric disaccharides at once, and up to three sequential stages of MSn in tandem, for the quantitative analysis of the relative percentage of each of these isomers. The isomer quantification was validated using mock mixtures and showed acceptable accuracy and precision. These methods may be applied to the quantification of other isomers by MSn. While each of the 12 disaccharides alone had a linear response to an internal standard in the MS1 spectra, the individual response factors did not remain constant when the concentrations of the other 11 disaccharides in the mixtures fluctuated, due to competition for electrospray ionization. The absolute concentration of one fluctuating isomer was determined out of a constant mixture of the other disaccharides. The rapid, accurate, and sensitive quantification of all isomeric disaccharides may contribute to the eventual sequencing of longer saccharides by MSn, enabling the elucidation of the structure-function relationships of HSGAGs.

Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling

“…GRIL-LC/MS is also a significant enhancement over LC/MS procedures that rely on linear equations to correlate the concentration of underivatized disaccharides with the detection of molecular standards (10,17,18,20). The presence of non-GAG contaminants, which is unavoidable in many samples, can affect analyte ionization and the calculated results.…”

“…To circumvent this problem, disaccharides bearing free amino groups were amidated (supplemental Fig. S4A) with propionic anhydride (20), which results in stronger interaction with the C18 matrix and separation from salts in the sample. Propionylation generates a product (D0R0) with a mass 56 atomic mass units larger than the corresponding free amino disaccharide (supplemental Fig.…”

“…Although the use of LC/MS has greatly enhanced disaccharide analysis, the technology suffers in one important aspect: the technique is semiquantitative due to variation in the ionization efficiency of different disaccharides, solvent effects on ionization, and suppression by contaminants in the preparation. To circumvent these problems, LC/MS procedures have been developed that utilize linear equations to correlate the concentration of underivatized disaccharides with the detection of molecular standards (10,(17)(18)(19)(20). Another approach is to use mass-tagging techniques based on differentially isotopically labeled tags introduced into oligosaccharides derived from intact chains (16,(21)(22)(23)(24).…”

Glycosaminoglycans in Human and Bovine Serum: Detection of Twenty-Four Heparan Sulfate and Chondroitin Sulfate Motifs Including a Novel Sialic Acid-Modified Chondroitin Sulfate Linkage Hexasaccharide

“…[26][27][28][29][30][31][32][33][34][35][36][37][38][39] Electrospray ionization mass spectrometry (ESI MS) utilizes a flowing stream containing the analyte, provides for very gentle ionization, and detects all ions simultaneously. Reverse phase HPLC coupled with ESI-MS greatly benefits GAG disaccharide analysis.…”

Effects of sulfate position on heparin octasaccharide binding to CCL2 examined by tandem mass spectrometry

“…One issue of concern is the liability of the sulfate groups on these molecules, however solvent conditions can be manipulated to minimize this loss [20,21]. Complete enzymatic digestion to disaccharide subunits for compositional analysis overcomes the problem of sulfate loss, but has the disadvantage that any information about uronic acid epimerization or sequence is lost [6,22]. Methods using partial digestion, isotopic labeling, and LC-MS have been developed [23][24][25][26], and these studies have helped to propel forward the field of GAG analysis.…”