Pavek John G, Whitworth Isabella T, Nakayama Lisa, Scalf Mark, Frey Brian L, Smith Lloyd M
Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave. Madison, Wisconsin 53706, United States.
J Proteome Res. 2025 Jan 3;24(1):323-332. doi: 10.1021/acs.jproteome.4c00838. Epub 2024 Dec 11.
Top-down proteomics, the characterization of intact proteoforms by tandem mass spectrometry, is the principal method for proteoform characterization in complex samples. Top-down proteomics relies on precursor isolation and subsequent gas-phase fragmentation to make proteoform identifications. While this strategy can produce highly detailed molecular information, the reliance on time-intensive tandem MS limits the speed with which proteoforms can be identified. We suggest that once proteoforms have been identified by top-down analysis in a system of interest, and archived in a system-specific Proteoform Atlas, subsequent analyses in that system can utilize the Atlas information to enable simpler and faster MS1-only identifications. We explore this idea here, using the ribosome as a model system of limited complexity. We used deep top-down analysis to construct an ribosomal Proteoform Atlas containing 2099 proteoforms from 52 of the 54 proteins that make up the ribosome. We show that using the Atlas enables confident MS1-only identifications of ribosomal proteoforms from that were perturbed by exposure to cold. Furthermore, this Atlas strategy identifies proteoforms up to 77% more rapidly compared to top-down identifications that require acquisition of both MS1 and MS2 spectra.
自上而下的蛋白质组学，即通过串联质谱对完整蛋白质异构体进行表征，是复杂样品中蛋白质异构体表征的主要方法。自上而下的蛋白质组学依靠前体离子分离及随后的气相碎裂来鉴定蛋白质异构体。虽然这种策略可以产生高度详细的分子信息，但对耗时的串联质谱的依赖限制了蛋白质异构体的鉴定速度。我们认为，一旦通过自上而下的分析在感兴趣的系统中鉴定出蛋白质异构体，并将其存档于特定系统的蛋白质异构体图谱中，那么在该系统中的后续分析就可以利用图谱信息来实现仅通过一级质谱（MS1）进行更简单、快速的鉴定。我们在此以核糖体作为复杂度有限的模型系统来探讨这一想法。我们使用深度自上而下分析构建了一个核糖体蛋白质异构体图谱，其中包含构成核糖体的54种蛋白质中52种的2099种蛋白质异构体。我们表明，使用该图谱能够仅通过一级质谱就可靠地鉴定出因暴露于低温而受到扰动的核糖体蛋白质异构体。此外，与需要同时采集一级质谱和二级质谱（MS2）图谱的自上而下鉴定相比，这种图谱策略鉴定蛋白质异构体的速度快了多达77%。
