An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies
An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies
复制标题
DOI：
10.1002/pmic.201300366
复制
发表时间：
2014-05-01
期刊：
影响因子：
3.4
通讯作者：
Pasa-Tolic, Ljiljana
中科院分区：
文献类型：
--
作者：
Dekker, Lennard;Wu, Si;Pasa-Tolic, Ljiljana
我们先前已经表明，暴露于相同抗原的不同个体产生在其互补决定区（CDR）中具有相同突变的抗体，这表明CDR胰蛋白酶肽可以作为疾病诊断和预后的生物标志物。来源于疾病特异性抗体的完整Fab具有更高的潜力;它们可以潜在地用于疾病治疗，并且需要鉴定抗体所针对的抗原。然而，通过胰蛋白酶肽的LC-MS分析（即自下而上）的完整Fab序列表征已被证明对于抗体的混合物是不切实际的。为了应对这一挑战，我们开发了一种集成的自下而上和自上而下的MS方法，采用二维色谱法与傅里叶变换质谱法（FTMS）相结合，并应用这种方法对两种药物单克隆抗体的可变部分进行全面表征，其灵敏度与自下而上的标准相当。这些努力代表了鉴定患者样本中疾病特异性抗体的重要一步，具有潜在的重大临床影响。
We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens toward which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step toward the identification of disease specific antibodies in patient samples with potentially significant clinical impact.