Konno Ryo, Ishikawa Masaki, Nakajima Daisuke, Inukai Kaori, Ohara Osamu, Kawashima Yusuke
Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.
Department of Pediatrics, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
DNA Res. 2025 Jul 4;32(4). doi: 10.1093/dnares/dsaf019.
Proteomics using mass spectrometry (MS) has significantly advanced, offering deep insights into complex proteomes. The timsTOF MS platform with its parallel accumulation-serial fragmentation (PASEF) technology has achieved high scan speeds and high-quality spectra. Bruker's timsTOF HT, which features TIMS-XR technology, offers an improved dynamic range and analysis depth, supporting high sample loadings. Moreover, various improvements to the data-independent acquisition method based on the PASEF technology (diaPASEF) have been reported. Despite these advancements, most high-level deep proteomic reports are based on the Orbitrap Astral and Orbitrap Exploris 480, and analytical systems using timsTOF MS still require improvement. Here, Bruker's timsTOF HT was used to validate and optimize key diaPASEF parameters, leading to the development of a Thin-diaPASEF method. This method provides a high quantitative accuracy and consistency. In our validation, 9,400 proteins were identified in a single shot from HEK cells (strictly controlled protein false discovery rate <1%), the highest number analysed by the timsTOF MS series using standard human cultured cells. Furthermore, by combining Thin-diaPASEF with an improved Lycopersicon esculentum lectin method, over 5,000 proteins were identified in a 24-sample/d analysis from the plasma, and we succeeded in constructing a system with high proteome coverage that can be used for biomarker discovery.
使用质谱（MS）的蛋白质组学取得了显著进展，能够深入洞察复杂的蛋白质组。配备平行累积-序列碎裂（PASEF）技术的timsTOF MS平台实现了高扫描速度和高质量的光谱。布鲁克的timsTOF HT采用了TIMS-XR技术，具有更高的动态范围和分析深度，支持高样本加载量。此外，基于PASEF技术的数据非依赖型采集方法（diaPASEF）也有了各种改进。尽管有这些进展，但大多数高级深度蛋白质组学报告仍基于Orbitrap Astral和Orbitrap Exploris 480，使用timsTOF MS的分析系统仍需改进。在此，使用布鲁克的timsTOF HT对关键的diaPASEF参数进行验证和优化，从而开发出一种Thin-diaPASEF方法。该方法具有很高的定量准确性和一致性。在我们的验证中，从HEK细胞单次分析中鉴定出9400种蛋白质（严格控制的蛋白质错误发现率<1%），这是使用标准人类培养细胞的timsTOF MS系列分析的最高数量。此外，通过将Thin-diaPASEF与改进的番茄凝集素方法相结合，在血浆24样本/天的分析中鉴定出超过5000种蛋白质，我们成功构建了一个具有高蛋白组覆盖率的系统，可用于生物标志物发现。
