Kurogochi Masaki, Amano Junko
Laboratory of Glycobiology, The Noguchi Institute, 1-8-1, kaga, Itabashi, Tokyo 173-0003, Japan.
Molecules. 2014 Jul 9;19(7):9944-61. doi: 10.3390/molecules19079944.
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.
我们开发了一种使用稳定同位素标记和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行定量糖肽谱分析的有效且灵敏的方法。在本研究中，我们合成了苯甲酸-d0 N-琥珀酰亚胺酯（BzOSu）和苯甲酸-d5 N-琥珀酰亚胺酯（d-BzOSu）作为轻、重同位素试剂，用于稳定同位素定量，以对糖肽进行比较分析。使用这种方法通过MALDI-TOF MS在正、负离子模式下均提高了电离效率。这些试剂与来自人血清IgG（hIgG）的糖肽在广泛的浓度范围内进行定量反应；糖肽的标记效率显示出高重现性，并获得了良好的校准曲线。为了证明这种方法的实际效用，我们对来自hIgG和骨髓瘤血浆产生的IgG1的糖肽结构进行了表征。通过将Bz标记的IgG1糖肽与d-Bz标记的hIgG糖肽混合对糖肽进行定量分析。通过将化脓性链球菌（endoS）或肺炎链球菌（endo-D）的内切β-N-乙酰氨基葡萄糖苷酶的高度特异性识别与MALDI-TOF MS分析相结合，证明了hIgG糖肽的聚糖结构鉴定。所获得的数据揭示了IgG1、IgG2和IgG3糖肽上含有半乳糖基化延伸的聚糖谱和聚糖结构异构体的比例。
