Partial enzymatic reactions: A missed opportunity in proteomics research.
Partial enzymatic reactions: A missed opportunity in proteomics research.
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DOI：
10.1002/rcm.8283
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发表时间：
2018-12-15
期刊：
影响因子：
--
通讯作者：
Lazar IM
中科院分区：
文献类型：
--
作者：
Deng J;Julian MH;Lazar IM
生物学研究依靠蛋白质组学工作流程以不断增长的速度进行。虽然MS数据采集高度自动化和快速，但样品制备仍然是开发高通量工作流程的瓶颈。特别是酶促蛋白质加工，涉及耗时的方案，可以从一天延长到另一天。为了解决这一差距，我们开发和评估了简单的，在几分钟内展开的溶液中胰蛋白酶酶促反应，并证明了该方法用于快速分析癌细胞提取物中蛋白质的实用性。胰蛋白酶酶促反应进行7-60 min，并将结果与进行18 h的常规方法的结果进行比较。相对于18小时程序，没有改变其他反应条件。通过nano-HPLC/MS/MS分析反应产物，并根据肽/蛋白质鉴定、序列覆盖率、肽长度、缺失切割位点、生成离子的质量和肽亲水/疏水性质评估产物的质量。结果表明，简短的，因此不完整的，酶促过程导致大量的肽片段，提高蛋白质序列和蛋白质组的覆盖率，从这些肽产生的串联质谱是高质量的可靠的蛋白质鉴定，和肽的物理性质是倾向于支持替代的多维分离和中间向下的蛋白质组学分析策略的发展。在酶消化的几分钟内产生相同肽的再现性非常接近于从18小时长的反应中获得的再现性，并且短反应和长反应的组合结果使蛋白质组覆盖率增加约40%。我们证明，在短时间尺度上进行的部分酶促反应是蛋白质组学研究的宝贵资产，并提出其实施简单，成本效益高，独立的协议，大大简化了生物样品的分析，或作为补充协议，提高蛋白质序列和蛋白质组覆盖率。
Biological studies are conducted at ever increasing rates by relying on proteomic workflows. Although MS data acquisition is highly automated and rapid, sample preparation continues to be the bottleneck of developing high-throughput workflows. Enzymatic protein processing, in particular, involves time-consuming protocols that can extend from one day to another. To address this gap, we developed and evaluated simple, in-solution tryptic enzymatic reactions that unfold within a few minutes, and demonstrate the utility of the methodology for the rapid analysis of proteins originating from cancer cell extracts. Tryptic enzymatic reactions were conducted for 7–60 min, and the results were compared to that of a routine approach conducted for 18 h. No other reaction conditions were changed relative to the 18 h procedure. The reaction products were analyzed by nano-HPLC/MS/MS, and the quality of the products was assessed in terms of peptide/protein identifications, sequence coverage, peptide length, missed-cleavage sites, quality of generated ions, and peptide hydrophilic/hydrophobic properties. The results demonstrate that brief, and therefore incomplete, enzymatic processes lead to a large number of peptide fragments that improve protein sequence and proteome coverage, that the tandem mass spectra produced from these peptides are of high quality for reliable protein identifications, and that the physical properties of peptides are prone to supporting the development of alternative multi-dimensional separations and middle-down proteomics analysis strategies. The reproducibility of generating the same peptides within a few minutes of enzymatic digestion was remarkably close to that obtained from 18 h long reactions, and the combined results of short and long reactions increased proteome coverage by ~40 %. We demonstrate that partial enzymatic reactions conducted on short time-scales represent a valuable asset to proteomic studies, and propose their implementation either as simple, cost-effective, stand-alone protocols for substantially streamlining the analysis of biological samples, or as complementary protocols, for improving protein sequence and proteome coverage.
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影响因子：
1.2
作者：
Lazar，Iulia M.;Deng，Jingren;Smith，Nicole
通讯作者：
Smith，Nicole
影响因子：
14.9
作者：
Vizcaíno JA;Csordas A;del-Toro N;Dianes JA;Griss J;Lavidas I;Mayer G;Perez-Riverol Y;Reisinger F;Ternent T;Xu QW;Wang R;Hermjakob H
通讯作者：
Hermjakob H
影响因子：
48
作者：
Wu, Cong;Tran, John C.;Zamdborg, Leonid;Durbin, Kenneth R.;Li, Mingxi;Ahlf, Dorothy R.;Early, Bryan P.;Thomas, Paul M.;Sweedler, Jonathan V.;Kelleher, Neil L.
通讯作者：
Kelleher, Neil L.
影响因子：
4.1
作者：
Yuan, Huiming;Zhang, Lihua;Zhang, Yukui
通讯作者：
Zhang, Yukui
影响因子：
4.4
作者：
Swaney, Danielle L.;Wenger, Craig D.;Coon, Joshua J.
通讯作者：
Coon, Joshua J.