Reliable LC‐MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards

Abstract

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated Nglycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments.

Recent advances in mass spectrometric analysis of glycoproteins

“…Recently, a stable isotope‐labeled IgG2b mAb, iGlycoMab, has been used as an internal standard to minimize errors caused by sample handling (digestion and derivatization) and instrumentation (fluctuation of nanoESI efficiency) in N‐glycan profiling by LC–MS analysis . Although, the unexpected degradation of isotopically labeled glycans was observed when iGlycoMab was denatured and digested with blood serum, the stability of the labeled glycan was improved by increasing the denaturation temperature and by performing a tryptic digest of the samples prior to the addition of iGlycoMab internal standard.…”

Advances in mass spectrometry‐based glycomics

“…A) and normalized data using iGlycoMab (Fig. B) . Moreover, the decrease in deviation of replicate analysis was observed with complex biological samples such as a cell line for both abundant and less abundant N‐ glycans.…”

Recent Advances in the Analysis of Complex Glycoproteins

“…120 Absolute quantification can also be facilitated by using an internal “unnatural” glycan standard which is used to spike all samples which are analyzed with MS, equally facilitating normalization of the MS signal in different analytical experiments. 72 …”