Proteins derived by recombinant technologies must be characterized to ensure quality, consistency and optimal production. These properties are usually assayed following purification procedures that are time consuming and labor intensive. Here, we describe a native mass spectrometry (MS) approach, direct-MS, for rapid characterization of intact overexpressed proteins immediately from crude samples. In this protocol, we discuss the multiple applications of the method and outline the necessary steps required for sample preparation, data collection and interpretation of results. We begin with the sample preparation workflows, which are relevant for recombinant proteins produced within bacteria, those analyzed straight from crude cell lysate, and secreted proteins generated in eukaryotic expression systems that are assessed directly from the growth culture medium. We continue with the mass acquisition steps that enable immediate definition of properties such as expressibility, solubility, assembly state, folding, overall structure, stability, post-translational modifications and associations with biomolecules. We demonstrate the applicability of the method by presenting the characterization of a computationally designed toxin–antitoxin heterodimer, activity and protein-interaction determination of a regulatory protein and detailed glycosylation analysis of a designed intact antibody. Overall, we describe a simple and rapid protocol that is relevant to both prokaryotic and eukaryotic expression systems and can be carried out on multiple mass spectrometers, such as Orbitrap and quadrupole time-of-flight (QTOF)-based mass spectroscopy platforms, that enable intact protein detection. The procedure takes from 30 min to several hours, from sample collection to data acquisition, depending on the depth of MS analysis.

必须对重组技术衍生的蛋白质进行表征，以确保质量、一致性和最佳生产。这些特性通常在费时费力的纯化程序之后进行测定。在这里，我们描述了一种原生质谱 (MS) 方法，直接 MS，用于立即从粗样品中快速表征完整的过表达蛋白质。在本协议中，我们讨论了该方法的多种应用，并概述了样品制备、数据收集和结果解释所需的必要步骤。我们从样品制备工作流程开始，这些工作流程与细菌中产生的重组蛋白相关，这些重组蛋白直接从粗细胞裂解物中进行分析，以及在真核表达系统中产生的分泌蛋白，这些蛋白直接从生长培养基中进行评估。我们继续进行质量采集步骤，这些步骤能够立即定义特性，例如可表达性、溶解度、组装状态、折叠、整体结构、稳定性、翻译后修饰以及与生物分子的关联。我们通过展示计算设计的毒素-抗毒素异二聚体的表征、调节蛋白的活性和蛋白质相互作用测定以及设计的完整抗体的详细糖基化分析来证明该方法的适用性。总体而言，我们描述了一种与原核和真核表达系统相关的简单快速的协议，并且可以在多个质谱仪上进行，例如基于 Orbitrap 和四极杆飞行时间 (QTOF) 的质谱平台，可实现完整蛋白质的检测。该过程需要 30 分钟到几个小时，从样品收集到数据采集，具体取决于 MS 分析的深度。