Quantitative glycomics strategies

Abstract

The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed.

Fig. 1.

CE-LIF and LC-fluorescence detection quantitative glycomic strategies.

Fig. 2.

Quantitative glycomics of stable isotopically permethylated glycans via ESI-, MALDI-, and LC-ESI-MS.

Fig. 3.

Quantitative glycomics incorporating labeling with stable isotopic reagents. A, labeling with (d0/d4) 2-AA. Reproduced and modified from Ref. with permission. B, labeling with [¹²C₆]aniline and [¹³C₆]aniline. Reproduced and modified from Ref. with permission. C, labeling via reductive amination using tetraplex tagging reagents. Reproduced and modified from Ref. with permission. D, labeling with stable isotope hydrazide reagents. Reproduced and modified from Ref. with permission. E, labeling with isobaric tandem mass tag reagents. Reproduced and modified from Ref. with permission.

Fig. 4.

A, Isotopic detection of aminosugars with glutamine (IDAWG) labeling theory and strategy. A schematic of the hexosamine biosynthetic pathway that converts the glycolysis intermediate fructose-6-phosphate (Fru-6-P) to UDP-GlcNAc showing the addition of nitrogen into the pathway from the amide side chain of Gln. UDP-GlcNAc goes on to generate UDP-GalNAc and CMP-Neu5Ac so that the aminosugars GlcNAc (blue square), GalNAc (yellow square), or Neu5Ac (purple diamond) in glycoconjugates will have an additional +1 Da added to their mass if amide-15N-Gln is used as the nitrogen donor. Glc, glucose; GlcN, glucosamine. Schematic of the IDAWG in vivo cell culture labeling strategy for comparative glycomics. Amide-15N-Gln or Gln is added to the media to label cultured cells. Upon harvesting, cells can be combined, and then N-linked and O-linked glycans can be isolated from digested proteins for permethylation and tandem mass spectrometry analysis. B, isotope pattern of light and heavy Man7GlcNAc2. Reproduced and modified from Ref. with permission from the publisher.