Data-Independent Acquisition Protease-Multiplexing Enables Increased Proteome Sequence Coverage Across Multiple Fragmentation Modes.
Data-Independent Acquisition Protease-Multiplexing Enables Increased Proteome Sequence Coverage Across Multiple Fragmentation Modes.
复制标题
DOI：
10.1021/acs.jproteome.1c00960
复制
发表时间：
2022-04-01
影响因子：
4.4
通讯作者：
Swaney, Danielle L.
中科院分区：
文献类型：
--
作者：
Richards, Alicia L.;Chen, Kuei-Ho;Wilburn, Damien B.;Stevenson, Erica;Polacco, Benjamin J.;Searle, Brian C.;Swaney, Danielle L.
多种蛋白酶的使用已被证明可以增加蛋白质组学实验中的蛋白质序列覆盖率，但由于需要额外的分析时间，它尚未在常规数据依赖采集 (DDA) 蛋白质组工作流程中广泛采用。或者，数据独立采集 (DIA) 具有分析来自不同蛋白酶消化的多重样品的潜力，但主要针对胰蛋白酶肽片段化进行了优化。在这里，我们评估了将三种蛋白水解消化物（胰蛋白酶、AspN 和 GluC）组合到单个样品中的 DIA 多重方法。我们首先使用规范的 DIA 片段化模式（光束类型 CID）以及共振激发 CID 分别优化每种蛋白酶的数据采集条件，以确定蛋白酶的最佳一致条件。接下来，我们证明，与单独使用胰蛋白酶相比，将这些条件应用于蛋白酶复合的人类肽样品会产生相似的蛋白质鉴定和定量性能，但肽检测量增加高达 63%，非冗余氨基酸检测量增加 45%。非胰蛋白酶肽能够实现非规范蛋白质同工型测定，并导致多种蛋白质的 100% 序列覆盖率，表明该方法在序列覆盖度至关重要的应用中有用，例如蛋白质同工型分析。
The use of multiple proteases has been shown to increase protein sequence coverage in proteomics experiments, however due to the additional analysis time required, it has not been widely adopted in routine data-dependent acquisition (DDA) proteomic workflows. Alternatively, data-independent acquisition (DIA) has the potential to analyze multiplexed samples from different protease digests, but has been primarily optimized for fragmenting tryptic peptides. Here we evaluate a DIA multiplexing approach that combines three proteolytic digests (Trypsin, AspN, and GluC) into a single sample. We first optimize data acquisition conditions for each protease individually with both the canonical DIA fragmentation mode (beam type CID), as well as resonance excitation CID, to determine optimal consensus conditions across proteases. Next, we demonstrate that application of these conditions to a protease-multiplexed sample of human peptides results in similar protein identifications and quantitative performance as compared to trypsin alone, but enables up to a 63% increase in peptide detections, and a 45% increase non-redundant amino acid detections. Non-tryptic peptides enabled non-canonical protein isoform determination and resulted in 100% sequence coverage for numerous proteins, suggesting the utility of this approach in applications where sequence coverage is critical, such as protein isoform analysis.
登录
查看更多内容
影响因子：
9.9
作者：
通讯作者：
--
影响因子：
8.8
作者：
Low, Teck Yew;van Heesch, Sebastiaan;Guryev, Victor
通讯作者：
Guryev, Victor
影响因子：
4.4
作者：
Cox, Juergen;Neuhauser, Nadin;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
4.6
作者：
Nolte H;MacVicar TD;Tellkamp F;Krüger M
通讯作者：
Krüger M