Department of Chemistry, The Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Proteomics. 2010 Oct;10(20):3589-97. doi: 10.1002/pmic.201000177.
Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale.
将高通量自上而下 MS 应用于整个蛋白质组需要一个尚未建立的数据处理模型。由于自上而下的方法在大规模上变得可行，我们报告了我们最新的软件管道，旨在以自动化的方式捕捉完整蛋白质数据的全部价值。对于完整质量的检测，我们结合了用于处理来自同位素解析（FT）和电荷状态解析（离子阱）LC-MS 数据的 MS1 数据的算法，然后将其与片段离子链接，以便使用 ProSight 进行数据库搜索。这是一个确定人类角蛋白和微管蛋白同工型的结果。针对整个蛋白质的复杂性进行了优化，新的软件模块基于 LC 保留时间和完整质量的强度可视化蛋白质组规模的数据，并能够选择性地检测 PTM，以自动筛选乙酰化、磷酸化和甲基化。除了正在经历化学应激的人类细胞外，我们还使用来自酵母菌株的比较 LC-MS 数据证明了软件功能。我们进一步推进这些进展，作为在蛋白质组学规模上实现自上而下 MS 的关键方面。
