Directed sample interrogation utilizing an accurate mass exclusion-based data-dependent acquisition strategy (AMEx).
Directed sample interrogation utilizing an accurate mass exclusion-based data-dependent acquisition strategy (AMEx).
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DOI：
10.1021/pr801017a
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发表时间：
2009-06
影响因子：
4.4
通讯作者：
Jaffe JD
中科院分区：
文献类型：
--
作者：
Rudomin EL;Carr SA;Jaffe JD
当使用质谱分析表征复杂的蛋白质组混合物时，进行彻底采样的能力至关重要。一种常见的方法是通过 LC-MS/MS 多次重新询问样品。然而，蛋白质组学研究中通常使用的传统数据依赖型采集方法通常会对高强度前体离子进行冗余采样，而无法完全对低强度前体离子进行采样。我们描述了一种方法，其中成功鉴定的肽的质量用于生成准确的质量排除列表，以便在后续分析期间不选择那些前体进行测序。我们使用基于精确质量排除的数据依赖型采集 (AMEx) 或标准数据依赖型采集进行了多次串联分析运行，以对复杂的细胞裂解物进行采样，结果发现，相对于标准 DDA 方法，在 ESI-Orbitrap 仪器上使用 AMEx 显着增加了经过验证的肽鉴定总数。额外鉴定的肽代表在样品中表现出低信号强度的前体离子。增加肽鉴定的总数增加了鉴定的蛋白质的数量，并提高了这些蛋白质的序列覆盖率。总之，这些数据表明使用 AMEx 是改善复杂蛋白质组混合物表征的有效策略。
The ability to perform thorough sampling is of critical importance when using mass spectrometry to characterize complex proteomic mixtures. A common approach is to re-interrogate a sample multiple times by LC-MS/MS. However, the conventional data-dependent acquisition methods that are typically used in proteomics studies will often redundantly sample high-intensity precursor ions while failing to sample low-intensity precursors entirely. We describe a method wherein the masses of successfully identified peptides are used to generate an accurate mass exclusion list such that those precursors are not selected for sequencing during subsequent analyses. We performed multiple concatenated analytical runs to sample a complex cell lysate, using either accurate mass exclusion-based data-dependent acquisition (AMEx) or standard data-dependent acquisition, and found that utilization of AMEx on an ESI-Orbitrap instrument significantly increases the total number of validated peptide identifications relative to a standard DDA approach. The additional identified peptides represent precursor ions that exhibit low signal intensity in the sample. Increasing the total number of peptide identifications augmented the number of proteins identified, as well as improved the sequence coverage of those proteins. Together, these data indicate that using AMEx is an effective strategy to improve the characterization of complex proteomic mixtures.
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影响因子：
7.4
作者：
Huang, JM;Zhang, P;Li, TY
通讯作者：
Li, TY
影响因子：
3.4
作者：
Scherl, A;Francois, P;Corthals, GL
通讯作者：
Corthals, GL
DOI：
10.1073/pnas.172170199
发表时间：
2002-08-20
影响因子：
11.1
作者：
Lipton, MS;Pasa-Tolic, L;Smith, RD
通讯作者：
Smith, RD
影响因子：
--
作者：
Andersson, KE
通讯作者：
Andersson, KE
影响因子：
46.9
作者：
Durr, E;Yu, JY;Schnitzer, JE
通讯作者：
Schnitzer, JE