PDF(Pubmed)
Abstract：
Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.
摘要：
蛋白质的重组表达，由治疗性抗体推动，已经发展成为一个价值数十亿美元的产业。这里至关重要的是关键属性的质量控制评估，如序列保真度，适当的折叠，和翻译后修饰(PTM)。错误会导致生物活性下降，在治疗性蛋白质的背景下，免疫原性风险升高。多年来,许多技术被开发并应用于以标准化和高通量的方式验证蛋白质。一个参数有，然而,到目前为止，很难评估。二硫化物桥,连接两个半胱氨酸残基的共价键,有助于蛋白质的正确折叠和稳定性，因此对其功效有重大影响。质谱有望成为以快速准确的方式发现它们的最佳技术。在这项工作中,我们提出了一种独特的样品制备组合，数据采集和分析有助于快速准确地评估纯化蛋白质中的二硫键。通过微波辅助酸水解（MAAH），蛋白质被快速消化，无伪影转化为肽，序列上有很大程度的重叠。这个程序的非特异性，然而,介绍了化学背景，通过在质谱测量之前对离子迁移率进行积分，可以有效地去除化学背景。消化步骤的非特异性还需要数据分析的新发展，为此，我们扩展了ProteomeDiscoverer（XlinkX/PD）中的XlinkX节点，以有效地处理数据并通过有效的错误发现率校正来确保正确性。整个工作流程可以在一小时内完成，允许高吞吐量，高精度的二硫键映射。
