When researchers evaluate protein function, one of the determinative aspects of structure that they turn to is glycosylation. This posttranslational modification affects over 60% of all cellular and membrane-bound proteins in humans. The identification and quantification of the carbohydrate attached to the protein during glycosylation, particularly the analysis of N-glycans, allows researchers to determine which particular structures result in specific functional activity. This information may offer insight into the progression and even treatment of conditions like certain cancers and inflammatory diseases that may derive biological functionality from specific glycan structure.
Unfortunately, glycoprotein analysis is difficult due to the varied structural nature of glycans, the high number of sites for glycosylation, and the abundance of glycan isomers. These challenges have led researchers to rely upon methods that require quantification and characterization of carbohydrates in multiple steps. Kalay et al.1 set out to compose a more streamlined approach that would allow for both accurate quantification and elaborate characterization of N-glycans within a single run on an LCQ decaXP ion trap mass spectrometer (Thermo Scientific).
The approach piloted by Kalay et al., referred to by the researchers as glycan nanoprofiling, involves the deglycosylation of N-glycans coupled with fluorophore tagging at the reducing end of the carbohydrate. The researchers then expose the glycans to concurrent electro-spray ionization mass spectrometry and nanofluorescence detection. The researchers use MS software to quantify each peak on the chromatogram and correlate it to a particular glycan structure.
Aside from the obvious benefit of combining these quantification and characterization protocols into a single run, the secondary benefit of glycan nanoprofiling is the potential for researchers to achieve high resolution analysis with a nanobore column that separates on the basis of both polarity and anionic charge, thus eliminating the need to perform both reverse phase separation and exoglycosidase treatments as in other methods. This is particularly useful when considering the abundance of glycan isomers, which share a molecular weight but whose divergent structures allow for differential elution through glycan nanoprofiling. An additional benefit of this method is that it eliminates the need to precalibrate the column. Kalay et al. found that coupling nanofluorescence with mass spectrometry resulted in single ion quantification and characterization with minimal background noise.
Kalay et al. also used glycan nanoprofiling to analyze the glycan structure of monoclonal antibodies with excellent correlative results when compared to traditional methods. The clinical implications of this test in the inflammatory properties of the antibodies, which researchers believe are specifically determined by the antibody’s glycan structure. This new method may have implications in the areas of diagnosis and treatment of certain cancers and autoimmune diseases. In terms of proteomics research, glycan nanoprofiling has the potential to allow researchers to quickly and accurately quantify and characterize glycan structures in the analysis of both purified proteins and intricate biological mixtures.
1. Kalay, H., et al. (2012) ‘Online nanoliquid chromatography-mass spectrometry and nanofluorescence detection for high-resolution quantitative N-glycan analysis‘, Analytical Biochemistry, 423 (2012), (pp. 153-162)