Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered, as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer: Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to, higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios Was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.