Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution.
Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution.
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DOI：
10.1021/jasms.2c00072
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发表时间：
2022-07-06
影响因子：
3.2
通讯作者：
Chen, Hao
中科院分区：
文献类型：
--
作者：
Ai, Yongling;Xu, Jeffrey;Gunawardena, Harsha P.;Zare, Richard N.;Chen, Hao
最近的研究表明，蛋白质的超快酶消化可以在250 μs内在微滴中实现。微滴消化（MD）产生的肽的进一步调查将是必要的，以评估它作为一种替代传统的散装消化自下而上和bioprotein蛋白质表征。在这里，我们研究和比较蛋白质胰蛋白酶消化MD和散装溶液。在β-乳球蛋白B的MD的情况下，由于消化时间短，观察到长肽的保存。此外，MD适用于消化混合物中的高丰度和低丰度蛋白质。在消化含有低水平常见宿主细胞蛋白（HCP）PLBL 2（mAb：PLBL 2 = 100：1（重量））的NIST 8671 mAb抗体的情况下，与过夜消化相比，MD产生的天冬酰胺/谷氨酰胺脱酰胺的消化诱导化学修饰水平较低。根据半切割和非特异性切割肽的检查，在胰蛋白酶消化特异性方面，MD和批量消化之间未观察到显著差异。我们的研究表明，MD，一种快速消化的方法，可以采用自下而上的蛋白质组学研究和单克隆抗体的肽图谱，以表征位点特异性脱酰胺和糖基化，用于生物药物的开发。
Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplet within 250 μs. Further investigation of peptides resulting from microdroplet digestion (MD) would be necessary to evaluate it as an alternative to the conventional bulk digestion for bottom-up and biotherapeutic protein characterization. Herein we examined and compared protein tryptic digestion in both MD and bulk solution. In the case of MD of β-lactoglobulin B, the preservation of long peptides was observed due to short digestion time. In addition, MD is applicable to digest both high- and low-abundance proteins in mixture. In the case of digesting NIST 8671 mAb antibody containing a low level of commonly encountered host cell protein (HCP) PLBL2 (mAb:PLBL2= 100:1 by weight), MD produced lower levels of digestion-induced chemical modifications of asparagine/glutamine deamidation, compared with overnight digestion. No significant difference between MD and bulk digestion was observed in terms of trypsin digestion specificity based on examination of semi- and unspecific-cleaved peptides. Our study suggests that MD, a fast digestion approach, could be adopted for bottom-up proteomics research and for peptide mapping of mAbs to characterize site-specific deamidation and glycosylation, for the purpose of development of biopharmaceuticals.
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影响因子：
8.4
作者：
Basuri P;Gonzalez LE;Morato NM;Pradeep T;Cooks RG
通讯作者：
Cooks RG
影响因子：
3.8
作者：
Gupta, Surbhi;Jiskoot, Wim;Rathore, Anurag S.
通讯作者：
Rathore, Anurag S.
影响因子：
5.8
作者：
Alves, Pedro;Arnold, Randy J.;Radivojac, Predrag
通讯作者：
Radivojac, Predrag
影响因子：
8.4
作者：
Bain, Ryan M.;Pulliam, Christopher J.;Cooks, R. Graham
通讯作者：
Cooks, R. Graham
影响因子：
15
作者：
Bark, SJ;Muster, N;Siuzdak, G
通讯作者：
Siuzdak, G