Quantification of Permethylated N-glycans derived from Human Blood Serum Using Multiple Reaction Monitoring (MRM) LC-MSMS

Glycosylation is the most prevalent posttranslational modification of proteins in mammalian cells, involved in modulating the activities and functions of proteins in health and disease.  Aberrant glycosylation has been recognized as the attribute of many mammalian diseases, including cancer.  Mass spectrometry has been utilized to compare glycomic profiles derived from different disease states.  Such quantitative comparison has been based on MALDI-MS and ESI-MS data.

Glycans were enzymatically released from glycoproteins: fetuin, alpha1-acid and ribonuclease B, and human blood serum using PNGase F. Glycans were purified using activated charcoal spin columns, permethylated using solid-phase permethylation.  Permethylated glycans were subjected to data-dependent LC-MSMS analysis using Velos Orbitrap hybrid mass spectrometry and then subjected to precursor ion scan and MRM (MS/MS) using Vantage TSQ triple-quadrupole mass spectrometry. 

Hydrophobicity induced by the permethylation of the glycan structures allowed for their efficient separation using capillary C18 columns.  This separation allowed for the detection of 81 glycan structures.  Tandem mass spectra acquired using Orbitrap hybrid MS were inspected for characteristic fragment ions specific for particular glycan structures.  A fragment ion at m/z value of 825 is only observed in the tandem mass spectra of sialylated glycans and fragment ion at m/z value of 420 is only observed in the tandem mass spectra of fucosylated glycans. Increased levels of glycan sialylation and fucosylation have been correlated with various cancers such as esophageal adenocarcinorma.  Reliable quantification of these structures using MRM LC/MSMS is the most reliable quantitative MS which will permit better means to monitor disease development and progression.