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Sequencing a Bispecific Antibody by Controlling Chain Concentration Effects When Using an Immobilized Nonspecific Protease.
Analytical Chemistry ( IF 7.4 ) Pub Date : 2020-06-29 , DOI: 10.1021/acs.analchem.0c01126
Robert A D'Ippolito 1 , Maria C Panepinto 1 , Keira E Mahoney 1 , Dina L Bai 1 , Jeffrey Shabanowitz 1 , Donald F Hunt 1, 2
Affiliation  

Complete sequence coverage of monospecific antibodies was previously achieved using immobilized aspergillopepsin I in a single LC-MS/MS analysis. Bispecific antibodies are asymmetrical compared to their monospecific antibody counterparts, resulting in a decrease in the concentration of individual subunits. Four standard proteins were used to characterize the effect of a decrease in concentration when using this immobilized enzyme reactor. Low concentration samples resulted in the elimination of large peptide products due to a greater number of enzymatic cleavages. A competitive inhibitor rich in arginine residues reduced the number of enzymatic cleavages to the protein and retained large molecular weight products. The digestion of a bispecific antibody with competitive inhibition of aspergillopepsin I maintained large peptide products better suited for sequence reconstruction, resulting in complete sequence coverage from a single LC-MS/MS analysis.

中文翻译:

当使用固定的非特异性蛋白酶时,通过控制链浓度效应对双特异性抗体进行测序。

以前在单个LC-MS / MS分析中使用固定的曲霉菌素I即可实现单特异性抗体的完整序列覆盖。双特异性抗体与其单特异性抗体对应物相比是不对称的,从而导致各个亚基浓度的降低。当使用这种固定化酶反应器时,使用四种标准蛋白质来表征浓度降低的影响。低浓度样品由于大量的酶促裂解而消除了大肽产物。富含精氨酸残基的竞争性抑制剂减少了对蛋白质的酶促切割次数,并保留了大分子量产物。
更新日期:2020-08-04
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