Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis.
Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis.
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在进行质谱分析之前，用于受控单克隆抗体消化的含胃蛋白酶的膜。
DOI：
10.1021/acs.analchem.5b02739
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发表时间：
2015-11-03
影响因子：
7.4
通讯作者：
Bruening, Merlin L.
中科院分区：
文献类型：
--
作者：
Pang, Yongle;Wang, Wei-Han;Reid, Gavin E.;Hunt, Donald F.;Bruening, Merlin L.
单克隆抗体（mab）是发展最快的一类治疗药物，因为它们对靶细胞具有高度特异性。治疗性单克隆抗体及其翻译后修饰（PTMs）的快速分析对于质量控制至关重要，质谱分析（MS）是抗体表征最强大的工具。本研究使用含有胃蛋白酶的尼龙膜作为受控蛋白水解反应器，在超高分辨率Orbitrap质谱分析之前进行单抗消化。抗体溶液在膜中的停留时间（从3 ms到3 s）的变化在不到10分钟内产生“自下而上”（1-2 kDa）到“中下”（5-15 kDa）的肽大小。这些肽覆盖了曲妥珠单抗和Waters抗体的整个序列，并且由来自Waters抗体的140个氨基酸组成的蛋白水解肽包含轻链上所有三个互补决定区域。这项工作比较了抗体轻链的“自下而上”（溶液内胰蛋白酶消化）、“自上而下”（完整蛋白质碎片化）和“中向下”（膜内消化）分析的性能。串联质谱的数据显示，“自下而上”、“自上而下”和“中向下”分析的键解理率分别为99%、55%和99%。膜内消化也有助于检测PTMs，如氧化、脱酰胺、n端焦谷氨酸形成和糖基化。与“自下而上”和“自上而下”的抗体鉴定方法相比，膜内酶切只需要最少的样品制备时间，并且该技术还可以获得高肽段和序列覆盖率，用于鉴定PTMs。
Monoclonal antibodies (mAbs) are the fastest growing class of therapeutic drugs, because of their high specificities to target cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. This study uses pepsin-containing nylon membranes as controlled proteolysis reactors for mAb digestion prior to ultrahigh-resolution Orbitrap MS analysis. Variation of the residence times (from 3 ms to 3 s) of antibody solutions in the membranes yields “bottom-up” (1–2 kDa) to “middle-down” (5–15 kDa) peptide sizes within less than 10 min. These peptides cover the entire sequences of Trastuzumab and a Waters antibody, and a proteolytic peptide comprised of 140 amino acids from the Waters antibody contains all three complementarity determining regions on the light chain. This work compares the performance of “bottom-up” (in-solution tryptic digestion), “top-down” (intact protein fragmentation), and “middle-down” (in-membrane digestion) analysis of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for “bottom-up”, “top-down”, and “middle-down” analyses, respectively. In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation, and glycosylation. Compared to “bottom-up” and “top-down” approaches for antibody characterization, in-membrane digestion uses minimal sample preparation time, and this technique also yields high peptide and sequence coverage for the identification of PTMs.
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影响因子：
3.8
作者：
Cai, Bing;Pan, Hai;Flynn, Gregory C.
通讯作者：
Flynn, Gregory C.
影响因子：
7.4
作者：
Fornelli, Luca;Ayoub, Daniel;Tsybin, Yury O.
通讯作者：
Tsybin, Yury O.
DOI：
10.1016/j.jchromb.2005.08.021
发表时间：
2005-11-05
影响因子：
3
作者：
Bailey, MJ;Hooker, AD;James, DC
通讯作者：
James, DC
影响因子：
48
作者：
Hahne, Hannes;Pachl, Fiona;Kuster, Bernhard
通讯作者：
Kuster, Bernhard
DOI：
10.1007/s13361-014-1015-8
发表时间：
2015-01-01
影响因子：
3.2
作者：
Long, Ying;Wood, Troy D.
通讯作者：
Wood, Troy D.