Characterization of the carbohydrate portion of glycoproteins in a high-throughput, cost-efficient manner presents myriad technological challenges. Unlike the linear peptide components of these molecules, carbohydrates can be complex, branched structures containing monomeric units that may link an oligosaccharide to a protein at more than one site.
Furthermore, a single sugar molecule can attach to multiple other sugars, and more than one oligosaccharide can attach to a single glycosylation site on a glycoprotein. Oligosaccharides can also contain a number of different sugar residues, some of which may have an identical mass, complicating molecular weight-based analyses.
“An oligosaccharide composed of three monomeric units can have 64,000 potential isomers,” said Fan Xiang, Ph.D., application development scientist at Shimadzu. All of these factors contribute to the complexity of glycosylation profiling, which encompasses a determination of the carbohydrate composition of a glycoprotein and the identity and structure of the various glycan isoforms.
Conventional HPLC and mass spectrometry (MS) technology have been sufficient for more qualitative analysis of the carbohydrate structures added on during post-translational modification (PTM) of a protein, but they have limitations when it comes to quantitative and structural analysis of glycosylation.
“Most groups doing glycan analysis only have MS2 capability,” explained Dr. Xiang, and they are “still struggling to see glycan structure.” Tandem mass spectrometry (MSn) techniques are increasingly being used to define detailed glycan structures—these include MS2 and MS3 approaches, and up to MS5 for more complex isoforms.
“Tandem mass spectrometric experiments have revealed that oligosaccharides might have characteristic signal-intensity profiles that depend on the glycosidic linkage and branching structures,” added Dr. Xiang. By developing an MS spectral library of structurally defined oligosaccharides, MS profiling strategies can then be used to compare the MSn spectra from an experimental analyte to the glycan spectral library, enabling identification of the carbohydrate structure and isomeric components using comparative peak analysis algorithms.
Dr. Xiang and colleagues at Shimadzu collaborated with researchers in the department of chemistry at the University of Michigan, Ann Arbor, on research presented at the ASMS meeting in Salt Lake City. They described the use of MALDI-QIT-TOF MS technology to determine the N-glycan structure of human CD24, a small glycosylphosphoinositol-anchored protein involved in signal transduction in T cells and immune-system activation. ]
The carbohydrate portion of CD24 and, in particular, the negatively charged sialic acid moieties on the sialylated N-glycan structures are believed to mediate protein-protein interactions involving CD24.
Deglycosylation of CD24 with PNGase F was followed by N-glycan purification on a porous graphitized carbon column. Half of the N-glycan fraction isolated was subjected to in-column permethylation. MALDI-QIT-TOF MS was used to analyze both the natural and permethylated N-glycan fractions, and the untreated fraction was analyzed in both positive and negative mode. Using this technique, the researchers identified seven carbohydrates, including three sialylated N-glycans.
From a drug development and regulatory perspective, glycosylation profiling presents additional challenges, noted Dr. Xiang, as there is no agreement or formal documentation on how to submit glycan structure data as part of a regulatory filing.