Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b
Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b
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DOI：
10.1016/j.ab.2010.08.033
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发表时间：
2011-01-01
影响因子：
2.9
通讯作者：
Pramanik, Birendra N.
中科院分区：
文献类型：
--
作者：
Liu, Yan-Hui;Wylie, David;Pramanik, Birendra N.
大肠杆菌衍生的重组人干扰素α-2b（rhIFN α-2b）的异构体Iso-2、Iso-3和Iso-4在发酵过程中通过蛋白质的翻译后修饰产生，在原料药的纯化和产量方面存在主要问题。我们在这里报告这些异构体的质谱（MS）方法的结构表征。对Iso-4（由高达75%的过程中IFN组成）和天然rhIFN α-2b进行了广泛的MS研究。通过液相色谱（LC）-MS分析两种样品产生的胰蛋白酶消化肽混合物，并通过LC-串联MS（三重四极杆质谱仪）、高分辨率MS（n）（LTQ Orbitrap）和基质辅助激光解吸/电离MS（MALDI-MS）进一步研究靶向肽。Iso-4的结构被阐明为IFN α-2b的N-末端半胱氨酸（Cysl）的新的葡糖酸酮亚胺衍生物，其中Cysl和Cys 98之间的二硫键被完全还原，而另一个二硫键对Cys 29-ss-Cys 138被部分还原。类似地，Iso-2被鉴定为在Cyst上乙酰化的正确二硫键折叠的rhIFN α-2b，Iso-3被鉴定为在Cysl上乙酰化的部分还原的rhIFN α-2b的S-谷胱甘肽化形式（Cys 98）。基于表征工作，成功实施了可重现的转化程序，将Iso-4转化为rhIFN α-2b。(C)2010年爱思唯尔公司All rights reserved.
The isoforms Iso-2, Iso-3, and Iso-4 of Escherichia colt-derived recombinant human interferon alpha-2b (rhIFN alpha-2b), generated by posttranslational modifications of the protein during fermentation, present a major problem in terms of purification and the yield of the drug substance. We report here the structural characterization of these isoforms by mass spectrometry (MS) methods. An extensive MS study was conducted on Iso-4, which is composed of up to 75% of the in-process IFN, and on the native rhIFN alpha-2b. The tlypsin-digested peptide mixtures generated from the two samples were analyzed by liquid chromatography (LC)-MS, and targeted peptides were further studied by LC-tandem MS (triple quadrupole mass spectrometer), high-resolution MS(n) (LTQ Orbitrap), and matrix-assisted laser desorption/ionization MS (MALDI-MS). The structure of lso-4 was elucidated as a novel pyruvic acid ketimine derivative of the N-terminal cysteine (Cysl) of IFN alpha-2b, where the disulfide bond between Cysl and Cys98 was fully reduced and the other disulfide bond pair, Cys29-ss-Cys138, was partially reduced. Similarly, Iso-2 was identified as a correctly disulfide-folded rhIFN alpha-2b with acetylation on Cyst, and Iso-3 was identified as an S-glutathionylated form (Cys98) of partially reduced rhIFN alpha-2b that was pyruvated on Cysl. Based on the characterization work, a reproducible conversion procedure was successfully implemented to convert Iso-4 to rhIFN alpha-2b. (C) 2010 Elsevier Inc. All rights reserved.