[개요]

단백질 내 분포하는 cysteine 중 disulfide-bond 를 형성하지 않는 free-thiol cysteine 에 대한 검출 및 정량 분석에 대한 응용 기술을 소개하는 논문입니다.

본 논문에서는 isotope-type의 Maleimide-PEG2-Biotin를 이용하는 방법을 활용하여 label-dependent detection 과 quantification을 진행하였으며 isotope type 이 아니더라도 N-ethylmaleimide 를 이용한 free-thiol(sulfhydryl) type의 cysteine 을 label 후 peptide mapping 분석을 통해 유용하게 detection 할 수 있는 분석기술이라 소개합니다.

[Abstract]

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-PEG2-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 d5-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).

KEYWORDS: Thiol, free sulfhydryl, NIST mAb, monoclonal antibody, mass spectrometry, cysteine, maleimide, disulfide

문헌 링크 : Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry (nih.gov)

관련 자료 : 분석서비스 > 분석게시판 > 분석서비스 소개 : Cysteine modification and detection scheme (emass.co.kr)

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