Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry

Abstract

An ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar “glyco tune mix”. When subjected to liquid chromatography–ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added 13C-galactose led to absolute quantification in the same experiment. Graphical AbstractDiscrepancy between desirable (grey bars) and real (green bars) relative ion abundance of equimolar amounts of glycans in positive mode ESI-MS. Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-017-0235-8) contains supplementary material, which is available to authorized users.

Protein–Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy

“…According to Schubert andc o-workers, serum albumin HSA-I from Sigma-Aldrich displays non-homogenous mixtures of two populations of sialic acids:t he a2,6-and a2,3-linked sialylgalactose residues as terminal sugars ( Figure S16). [32,33] Indeed, this point was confirmed also in the fractions of serum albumins under study,H SA-I and BSA-I (Figure S17 Aa nd S18). Nevertheless, our NMR analysis ( Figure S18 Aa nd B) showedt hat they contain more a2,6-linked sialylgalactose moieties than the a2,3-linked analogues in agreement with previous reported data.…”

“…In the presence of lactose, the effect on the line broadening, detected in the 1 H 15 N‐HSQC by addition of BSA‐I, was much less marked than that observed in absence of lactose (Figure S15 B and D), highlighting that BSA and lactose compete for the same galectin binding site. According to Schubert and co‐workers, serum albumin HSA‐I from Sigma–Aldrich displays non‐homogenous mixtures of two populations of sialic acids: the α2,6‐ and α2,3‐linked sialylgalactose residues as terminal sugars (Figure S16) . Indeed, this point was confirmed also in the fractions of serum albumins under study, HSA‐I and BSA‐I (Figure S17 A and S18).…”

“…Serum albumins (BSA‐I and HSA‐I) were selected due to their glycosylation content. Schubert and co‐workers have recently described that HSA‐I encodes a mixture of terminal sialic acids α2,6‐ and α2,3‐linked to β‐ d ‐galactose . Ficoll and PEG 3350 crowders were chosen as controls to address viscosity issues.…”

“…Schubert and co-workers have recently described that HSA-I encodes am ixture of terminal sialic acids a2,6-and a2,3-linked to b-d-galactose. [32,33] Ficoll and PEG 3350 crowders were chosen as controlst oa ddress viscosity issues. Ficoll is ap olymer formed by sugar (sucrose) units, whereas PEG is ar ather flexible polymer formed by ethylene ethers.…”

Reductive Alkaline Release of N‐Glycans Generates a Variety of Unexpected, Useful Products

“…For the preparation of quantities of labeled reference compounds involving unstable glycosylamines the chemical approach likewise can substitute the enzymatic route. The same can be said about reference glycans labeled by reductive amination or nonlabeled references for LC‐ESI‐MS . While it is tempting to suggest chemical release also for routine N‐glycan analysis with fluorescent dyes, the saving of enzyme costs will not justify the additional desalting step.…”