LC-MS/MS analysis glycans released from proteins can provide structural information such as monosaccharide composition, sequence, branching and linkages, which is important for an understanding of their role in biology. Three difficulties in characterising and quantifying these glycans is the high dynamic range of glycan structure abundance, the structural similarity between glycan isomers and the lack of a vendor-neutral, open-access tool for quantifying glycans across a large number of LC-MS/MS datasets. Here, a streamlined data acquisition and analysis platform for glycomics was applied to reduced N- and O-glycans released from membrane-bound and secreted glycoproteins extracted from drug treated neuronal cell lines.
Our data acquisition approach uses a porous graphitised carbon column to achieve liquid chromatographic separation of glycan structural isomers, allowing characteristic CID-MS/MS of each isomer to be acquired in negative mode. The use of a linear ion-trap mass spectrometer addresses the high dynamic range in glycan structural abundance by using an optimised instrument method to effectively characterise low abundance glycans. This method has provided characterisation of over seventy N-glycans on proteins secreted from neuronal cell lines. Glycan structural epitopes characterised include outer-arm fucosylation and oligo-sialic acid motifs which have previously been implicated in cancer and neuronal regeneration respectively.
The absence of an open access platform for label-free glycan quantification has an impact on data analysis throughput, reproducibility and data sharing. Using Skyline, a software tool used in targeted proteomics, ion chromatograms of each observed charge state of all characterised glycans were generated and corresponding peak areas were integrated automatically. The Skyline method was compared to our conventional manual analysis approach and high consistency was observed. The high throughput of this streamlined method was illustrated by the glycomics analysis of membrane-bound and secreted glycoproteins from a time-course study of human and mouse neuronal cell lines, investigating immune responses to lipopolysaccharide treatment.