Relative Quantitation of Glycopeptides Based on Stable Isotope Labeling Using MALDI-TOF MS

Abstract

Abstract:We have developed an effective, sensitive method for quantitative glycopeptide profiling using stable isotope labeling and MALDI-TOF mass spectrometry (MS). In this study, we synthesized benzoic acid-d 0 N-succinimidyl ester (BzOSu) and benzoic acid-d 5 N-succinimidyl ester (d-BzOSu) as light and heavy isotope reagents for stable isotope quantification for the comparative analysis of glycopeptides. Using this approach provided enhanced ionization efficiency in both positive and negative modes by MALDI-TOF MS. These reagents were quantitatively reacted with glycopeptides from human serum IgG (hIgG) at a wide range of concentrations; the labeling efficiency of the glycopeptides showed high reproducibility and a good calibration curve was obtained. To demonstrate the practical utility of this approach, we characterized the structures of glycopeptides from hIgG and from IgG1 produced by myeloma plasma. The glycopeptides were quantitatively analyzed by mixing Bz-labeled IgG1 glycopeptides with d-Bz-labeled hIgG glycopeptides. Glycan structural identification of the hIgG glycopeptides was demonstrated by combining the highly specific recognition of endo-β-N-acetyl glucosaminidases from Streptococcus pyogenes (endoS) or from Streptococcus pneumoniae (endo-D) with MALDI-TOF MS analysis. The obtained data revealed the glycan profile and the ratio of glycan structural isomers containing a galactosylated extension on IgG1, IgG2 and IgG3 glycopetides.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…Kurogochi and Amano () have synthesized benzoic acid‐d 0 and benzoic acid‐d 5 N ‐succinimidyl esters and shown that they provide enhanced ionization efficiency in both positive and negative modes by MALDI‐TOF MS. The difference of five mass units between the molecular weights avoided problems with isotopic interference from the d 0 derivatives.…”

Preparation and biological activities of anti-HER2 monoclonal antibodies with fully core-fucosylated homogeneous bi-antennary complex-type glycans

“…Glycopeptides were obtained from mAbs included in peak a of each sample by trypsin digestion, and their N-glycan structures were confirmed by LC-MS/MS. 23) The results showed that peaks a in panels (A), (B), (C), and (D) of Fig. 3 contain only N-glycans with G2F, G1aF, G1bF, and G0F structures, respectively.…”

“…15) Structures of N-glycans attached to the individual mAbs thus prepared were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using their glycopeptides as described. 15,23) A schematic illustration of preparation procedures as described above is shown in Supplement 4.…”

“…Relative amounts of transglycosylated products and untransglycosylated acceptors in each mAb sample. Glycopeptides were obtained from mAbs transglycosylated by Endo S variant and their N-glycan structures were confirmed by LC-MS/MS 23). Individual Nglycans are indicated with initials used inFig.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…However, as we discussed in a recent review (67), comparing the entire glycoform distribution (micro-heterogeneity) at individual glycosylation sites in separate conditions may be more informative in glycobiology. Both isotope-assisted quantitative glycoproteomics (46,105,108,164) and label-free (based on XIC area, precursor intensity or spectral count) quantitative glycoproteomics (21,104,106,159,165) can generate quantitative information of site micro-heterogeneity. Importantly, if the ambition is to understand the mechanisms driving alterations of the glycoproteome in specific conditions (see Fig.…”