A general protease digestion procedure for optimal protein sequence coverage and post-translational modifications analysis of recombinant glycoproteins: application to the characterization of human lysyl oxidase-like 2 glycosylation.
A general protease digestion procedure for optimal protein sequence coverage and post-translational modifications analysis of recombinant glycoproteins: application to the characterization of human lysyl oxidase-like 2 glycosylation.
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DOI：
10.1021/ac2017037
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发表时间：
2011-11-15
影响因子：
7.4
通讯作者：
Desaire, Heather
中科院分区：
文献类型：
--
作者：
Rebecchi, Kathryn R.;Go, Eden P.;Xu, Li;Woodin, Carrie L.;Mure, Minae;Desaire, Heather
利用重组DNA技术表达蛋白质治疗药物是药物研发的一个成熟领域。随着重组蛋白越来越多地被用作生物疗法，需要改进的方法来确保翻译后修饰(PTM)的特征。通常情况下，为PTM分析准备的蛋白质被蛋白质水解物消化并用质谱仪进行分析。为了确保对给定蛋白质的PTMS的完全覆盖，必须获得该蛋白质的完整序列覆盖，这通常是相当具有挑战性的。这项研究的目标是设计一种最大限度地扩大蛋白质序列覆盖率的方案，并能够检测翻译后修饰，特别是N-连接糖基化。为了实现这一目标，通过比较变性剂(尿素和Rapigest™SF)、还原剂(二硫苏糖醇和二硫苏糖醇和三(2-羧乙基)膦，TCEP)和不同浓度的烷化剂(碘乙酰胺，IAM)的优缺点，设计了一种高效的胰酶消化方法。用不同的酶消化法对人促转铁蛋白进行分析后，确定了较理想的条件：6M尿素用于变性，5 mM TCEP用于还原，10 mM IAM用于烷基化，10 mM DTT用于在添加胰酶之前冷却多余的IAM。将该方法成功地应用于一种新的重组蛋白--人赖氨酸类氧化物酶2(HLOXL2)。此外，在蛋白质中的两个糖基化位点很容易检测到糖基化的PTM。这些消化条件是专门为重组蛋白质和生物疗法的PTM分析而设计的，这里描述的工作填补了日益增长的生物药物分析领域的一个未得到满足的需求。
Using recombinant DNA technology for expression of protein therapeutics is a maturing field of pharmaceutical research and development. As recombinant proteins are increasingly utilized as biotherapeutics, improved methodologies ensuring the characterization of post-translational modifications (PTMs) are needed. Typically, proteins prepared for PTM analysis are proteolytically digested and analyzed by mass spectrometry. To assure full coverage of the PTMs on a given protein, one must obtain complete sequence coverage of the protein, which is often quite challenging. The objective of the research described here is to design a protocol that maximizes protein sequence coverage and enables detection of post-translational modifications, specifically N-linked glycosylation. To achieve this objective, a highly efficient proteolytic digest protocol using trypsin was designed by comparing the relative merits of denaturing agents (urea and Rapigest™ SF), reducing agents (dithiothreitol, DTT, and tris(2-carboxyethyl)phophine, TCEP), and various concentrations of alkylating agent (iodoacetamide, IAM). After analysis of human apo-transferrin using various protease digestion protocols, ideal conditions were determined to contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. This method was successfully applied to a novel recombinant protein, human lysyl oxidase-like 2 (hLOXL2). Furthermore, the glycosylation PTMs were readily detected at two glycosylation sites in the protein. These digestion conditions were specifically designed for PTM analysis of recombinant proteins and biotherapeutics, and the work described herein fills an unmet need in the growing field of biopharmaceutical analysis.
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影响因子：
4.4
作者：
Go, Eden P.;Chang, Qing;Liao, Hua-Xin;Sutherland, Laura L.;Alam, S. Munir;Haynes, Barton F.;Desaire, Heather
通讯作者：
Desaire, Heather
影响因子：
4.4
作者：
Arnold, RJ;Hrncirova, P;Novotny, MV
通讯作者：
Novotny, MV
影响因子：
2.9
作者：
Liu, Yan-Hui;Wylie, David;Pramanik, Birendra N.
通讯作者：
Pramanik, Birendra N.
HIV-1 疫苗开发中的挑战。
影响因子：
64.8
作者：
Barouch, Dan H.
通讯作者：
Barouch, Dan H.
DOI：
10.1016/s1570-9639(03)00053-0
发表时间：
2003-04-11
影响因子：
3.2
作者：
Molnar, J;Fong, KSK;Csiszar, K
通讯作者：
Csiszar, K