Department of Pharma Technical Development, Genentech, Oceanside, CA 92056, USA.
Anal Biochem. 2011 Oct 15;417(2):256-63. doi: 10.1016/j.ab.2011.06.024. Epub 2011 Jun 24.
We report a case study of characterization of a non-enzymatically glycated IgG1 using reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and mass spectrometry (MS). Glycation was found to occur nonspecifically at multiple sites in both the light and heavy chains. The glycated light and heavy chains result in wider peaks eluting late in the reducing CE-SDS profile; in particular, the glycated light chain behaved as a shoulder peak detected by either ultraviolet (UV) or laser-induced fluorescence (LIF) signals. The glycated species can be enriched by boronate affinity chromatography. Analyzing the enriched samples by reversed phase high-performance liquid chromatography in line with time-of-flight MS (RP-HPLC-TOF/MS) revealed adducts of +162 and +324 Da to both the light and heavy chains, suggesting the presence of multiple glycation sites. Tryptic peptide mapping and tandem mass sequencing were used to identify two glycation sites on each of the light and heavy chains.
我们报告了一例使用还原毛细管电泳十二烷基硫酸钠（CE-SDS）和质谱（MS）对非酶糖化 IgG1 进行特征描述的研究。在轻链和重链中，糖化均发生在多个非特异性部位。糖化的轻链和重链导致还原 CE-SDS 谱中晚期洗脱的峰变宽；特别是，糖化的轻链表现为紫外（UV）或激光诱导荧光（LIF）信号检测到的肩峰。糖化物质可以通过硼酸亲和层析进行富集。通过反相高效液相色谱与飞行时间质谱（RP-HPLC-TOF/MS）在线分析富集样品，发现轻链和重链上均有+162 和+324 Da 的加合物，表明存在多个糖化部位。胰蛋白酶肽图谱和串联质谱测序用于鉴定轻链和重链上的每个链上的两个糖化部位。
