Differential chemical derivatization integrated with chromatographic separation for analysis of isomeric sialylated N-glycans: a nano-hydrophilic interaction liquid chromatography-MS platform

Anal Chem. 2013 Sep 3;85(17):8421-8. doi: 10.1021/ac4018007. Epub 2013 Aug 19.

Abstract

MS analysis of sialylated glycans is challenging due to their low ionization efficiency in positive ion mode as well as the possibility of in-source fragmentation. Chemical derivatization strategies have been developed to address this issue focused on removal of the labile acidic proton prior to MS analysis. Highly sialylated negatively charged glycans also exhibit high retention and unsatisfactory separation efficiency when analyzed by hydrophilic interaction liquid chromatography (HILIC) due to their high polarity. Here, we combined linkage specific derivatization of sialic acids by reaction with the condensation reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) in methanol with nanoscale liquid chromatographic separation prior to accurate mass Orbitrap MS analysis. Coupling DMT-MM charge neutralization of sialic acids with nano-HILIC-Orbitrap-MS not only allows for linkage specific characterization of sialylated glycans directly from the precursor mass but also improves the preceding HILIC separation by increasing the hydrophobicity and altering the selectivity of the oligosaccharide analytes. We focused on the trisialylated N-glycan fraction from haptoglobin and human plasma, enriched using weak anion exchange chromatography, as this trisialylated fraction has been linked with cancer associated changes in the serum N-glycome. The developed methodology was applied to investigate whether structural alterations in this oligosaccharide pool, enriched from the sera of pathological stage and sex matched patients bearing lung, breast, ovarian, pancreatic, or gastric cancer, demonstrate any degree of cancer specificity or whether changes in expression levels are purely cancer associated. The results of this pilot study indicate limited degrees of cancer specificity, particularly for pancreatic cancer, based on alterations in the relative abundance of specific trisialylated isomers.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid / methods
  • Female
  • Humans
  • Isomerism
  • Nanotechnology / methods*
  • Polysaccharides / analysis*
  • Polysaccharides / chemistry*
  • Tandem Mass Spectrometry / methods*

Substances

  • Polysaccharides