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-d0 N-succinimidyl ester (BzOSu) and benzoic acid-d5 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.
Highlights
Glycosylation is one of the most ubiquitous and crucial processes in the posttranslational modification of proteins expressed in eukaryotic cells [1,2]
We synthesized benzoic acid-d0 N-succinimidyl ester (BzOSu) and benzoic acid-d5 N-succinimidyl ester (d-BzOSu) as light and heavy isotope mass tag reagents for stable isotope-based quantification in comparative glycoproteomics (Scheme 1). These compounds were functionalized with amine-reactive groups, and reacted with amine-containing glycopeptides to form an amide bond through a mild coupling reaction
The coupled glycopeptides could be detected under both positive and negative mode conditions in MALDI-TOF mass spectrometry (MS) due to their enhanced MS signal compared to unlabeled glycopeptides
Summary
Glycosylation is one of the most ubiquitous and crucial processes in the posttranslational modification of proteins expressed in eukaryotic cells [1,2]. The techniques used in glycoproteomics are becoming more sophisticated, allowing more detailed information to be obtained [4] The development of these techniques has led to clinical translational studies and its application to several fields, in particular to protein biomarker research. Stable isotopic or isobaric labeling using chemical reactions (ICAT and iTRAQ) [27,28], metabolic incorporation (SILAC) [29], enzymatic reactions (18O labeling [30]) and MRM-MS methods using ESI-triple-quadrupole MS instruments as label-free approaches [31] have been developed These techniques are generally based on LC-ESI-MS/MS systems with high separation performance. This study suggests that isotopic glycopeptide-labeling is a promising approach for quantitative glycopeptide profiling and holds significant potential for biological and clinical research
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