Xiang Tao, Chumsae Chris, Liu Hongcheng
Process Sciences Department, Abbott Bioresearch Center, 100 Research Drive, Worcester, Massachusetts 01605, USA.
Anal Chem. 2009 Oct 1;81(19):8101-8. doi: 10.1021/ac901311y.
A low percentage of free sulfhydryl is a common feature of recombinant monoclonal antibodies although, in theory, all cysteine residues should be involved in disulfide bonds. A differential alkylation method was developed to determine the percentage of free sulfhydryl at each cysteine residue of four recombinant monoclonal antibodies. Free sulfhydryl was first alkylated with 12C iodoacetic acid. Free sulfhydryl, resulting from the reduction of disulfide bonds, was then alkylated with 13C iodoacetic acid. Cysteine containing peptides that were modified by 13C iodoacetic acid showed a molecular weight that was 2 Da higher than the same peptide that was modified by 12C iodoacetic acid. Peptides, containing the same cysteine residues that were modified with both alkylation reagents, coeluted on reversed-phase chromatography. Analysis by mass spectrometry resulted in two partially overlapped m/z series for each cysteine containing peptide, corresponding to modification by iodoacetic acid with 12C or 13C. The percentage of free sulfhydryl was then calculated using the two m/z series at each cysteine site. A low percentage of free sulfhydryl was detected at every cysteine residue in the four antibodies studied. Although different antibodies contained different levels of free sulfhydryl, similar distribution of free sulfhydryl in the domain structures was observed in the four antibodies.
重组单克隆抗体的一个常见特征是游离巯基的比例较低，尽管从理论上讲，所有半胱氨酸残基都应参与二硫键的形成。开发了一种差异烷基化方法来测定四种重组单克隆抗体每个半胱氨酸残基的游离巯基百分比。首先用¹²C碘乙酸对游离巯基进行烷基化。然后，由二硫键还原产生的游离巯基用¹³C碘乙酸进行烷基化。用¹³C碘乙酸修饰的含半胱氨酸肽段的分子量比用¹²C碘乙酸修饰的同一肽段高2 Da。含有相同半胱氨酸残基且用两种烷基化试剂修饰的肽段在反相色谱上共洗脱。通过质谱分析，每个含半胱氨酸肽段产生两个部分重叠的m/z系列，分别对应于¹²C或¹³C碘乙酸的修饰。然后使用每个半胱氨酸位点的两个m/z系列计算游离巯基的百分比。在所研究的四种抗体的每个半胱氨酸残基处均检测到较低比例的游离巯基。尽管不同抗体的游离巯基水平不同，但在这四种抗体的结构域中观察到了相似的游离巯基分布。
