a Human Health Therapeutics Research Centre , National Research Council of Canada , Ottawa , Ontario , Canada.
MAbs. 2019 May/Jun;11(4):757-766. doi: 10.1080/19420862.2019.1595307. Epub 2019 Apr 16.
The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman's reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu™Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).
蛋白质中游离巯基的检测可以揭示不完全的二硫键形成情况，指示可供结合的半胱氨酸残基，并深入了解蛋白质的稳定性和结构。传统的游离巯基检测光谱方法，如 Ellman 试剂，通常需要相对大量的样品，这使得它们无法用于生物治疗药物在开发周期早期的分析。这些光谱方法也无法准确确定游离巯基的位置，进一步限制了它们的应用。本研究采用马来酰亚胺-PEG-生物素标记法检测完整蛋白质中的游离巯基残基，质谱法可以检测到 2%的游离巯基残基（每个蛋白质分子 0.02molSH）。还原后，可测量抗体重链和轻链上游离巯基的丰度。为了在肽水平分辨率上确定游离巯基的位置，分别用 N-乙基马来酰亚胺和 d-N-乙基马来酰亚胺对游离巯基和参与二硫键的半胱氨酸进行差异标记。酶切消化和纳升液相色谱-质谱分析后，可定量检测到每个半胱氨酸残基的游离巯基丰度低至 2%。该方法经过优化，可避免非特异性标记、二硫键重排、马来酰亚胺交换和水解。该游离巯基分析新工作流程用于测量 3 种市售单克隆抗体标准品（NIST 单克隆抗体参考材料（NIST）、SILu™Lite SigmaMAb 通用抗体标准品（Sigma-Aldrich）和完整 mAb 质量检查标准品（Waters））和 1 种小蛋白标准品（β-乳球蛋白 A）中游离巯基的含量和位置。
