A Sensitive and Controlled Data-Independent Acquisition Method for Proteomic Analysis of Cell Therapies

Mass spectrometry
(MS)-based proteomic measurements are uniquely
poised to impact the development of cell and gene therapies. With
the adoption of rigorous instrumental performance qualifications (PQs),
large-scale proteomics can move from a research to a manufacturing
control tool. Especially suited, data-independent acquisition (DIA)
approaches have distinctive qualities to extend multiattribute method
(MAM) principles to characterize the proteome of cell therapies. Here,
we describe the development of a DIA method for the sensitive identification
and quantification of proteins on a Q-TOF instrument. Using the improved
acquisition parameters, we defined a control strategy and highlighted
some metrics to improve the reproducibility of SWATH acquisition-based
proteomic measurements. Finally, we applied the method to analyze
the proteome of Jurkat cells that here serves as a model for human
T-cells. Raw and processed data were deposited in PRIDE (PXD029780).

基于质谱（Mass Spectrometry，MS）的蛋白质组学检测，在细胞与基因治疗的研发进程中拥有独特的应用前景。伴随严格的仪器性能确认（Performance Qualifications，PQs）规范的落地实施，大规模蛋白质组学分析可从科研工具升级为生产质控工具。其中，数据非依赖性采集（Data-independent Acquisition，DIA）技术尤为适配，其具备独特性能可拓展多属性方法（Multiattribute Method，MAM）的应用原理，以表征细胞治疗产品的蛋白质组。本文详细阐述了在Q-TOF质谱仪上实现蛋白质高灵敏度鉴定与定量的DIA方法开发流程。通过优化采集参数，我们确立了对应的质控策略，并明确了若干可提升基于SWATH采集的蛋白质组学检测重复性的关键指标。最后，本研究将所开发的方法应用于以Jurkat细胞作为人T细胞模型的蛋白质组分析。原始及处理后的实验数据已提交至PRIDE数据库（登录号：PXD029780）。