이매스교육 및 공지사항
[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
관련 자료 : 분석서비스 > 분석게시판 > 분석서비스 소개 : Cysteine modification and detection scheme (emass.co.kr)
저희 이매스 분석센터에서도 free-thiol 분석 서비스를 제공해드리고 있습니다.
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