In the released N-glycan analysis, N-glycans from a protein sample is cleaved out by the reaction with N-glycosidase F (PNGase F). The released glycan is then derivatized by the reaction with 2-aminopyridine. Optionally Sialidases is used to remove sialic residues from PNGase F cleavage. The 2-Ab labeled glycan species are analyzed by hydrophilic interaction liquid chromatography (HILIC) coupled with a fluorescence detector, as well online connected to a Tandem mass spectrometer. So three types of data are acquired from labeled glycan species: HPLC fluorescence profile, mass, and MS/MS fingerprint. The MS and MS/MS spectra give accurate and highly sensitive identification of each glycan species, while the result from HILIC gives a quantitative measurement of their ratio.
In site-specific N- and O-glycan analysis, multiple digestions are carried out on glycoprotein and resulted glycopeptides are analyzed by NanoLC-MS/MS. We have developed a specific bioinformatic software (Glycomap) which can recognize the MS pattern of different N- and O-glycopeptides. The determination of glycoforms at each specific glycosylation sites are further validated by our specialists.
The procedure for glycan linkage analysis is as following: first, glycan is released from glycoprotein by PNGase reaction, and chemically labeled with fluorescence tag 2-AB. The labeled glycan is reacted stepwise with different glycosidase step by step, with the removal of one terminal sugar from each step. The product from each step is analyzed by HILIC with a fluorescence detector and online coupled with a mass spectrometer.
For PTM analysis, we have developed a highly sensitive method based on NanoLC-MS/MS procedure that allows us to detect PTM at a low stoichiometry. Our PTMmap software is capable of detecting any known PTM. If possible, a pre-enrichment procedure is often carried out to enrich peptides or proteins that contain the PTM of interest. The determination of each modification is manually validated by specialists before reporting to clients.