Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics.
Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics.
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DOI：
10.1039/c7mb00393e
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发表时间：
2017-11-21
影响因子：
--
通讯作者：
Wu R
中科院分区：
文献类型：
--
作者：
Suttapitugsakul S;Xiao H;Smeekens J;Wu R
质谱（MS）已成为分析蛋白质的越来越重要的技术。在流行的自下而上的基于MS的蛋白质组学中，还原和烷基化是促进肽鉴定的常规步骤。但是，反应不完全和副反应可能会发生，这会影响实验结果。在这项工作中，我们系统地评估了常用试剂的还原步骤，即，二硫苏糖醇、2-巯基乙醇、三（2-羧乙基）膦或三（3-羟丙基）膦，和用碘乙酰胺、丙烯酰胺、N-乙基马来酰亚胺或4-乙烯基吡啶烷基化。通过使用来自酵母全细胞裂解物的消化肽，使用四种不同的还原试剂鉴定的蛋白质和肽的数量非常相似。然而，来自四种烷基化试剂的结果显著不同，其中碘乙酰胺得到最高数量的具有烷基化半胱氨酸的肽和最低数量的具有不完全半胱氨酸烷基化和副反应的肽。进一步优化了碘乙酰胺烷基化反应条件。为了鉴定更多的含半胱氨酸的多肽，采用硫丙基-Sepharose 6B树脂进行富集，并对还原和烷基化条件进行优化。富集导致含半胱氨酸的肽比不富集的多三倍以上。用不同试剂对还原和烷基化的系统评价可以帮助更好地设计自下而上的蛋白质组学实验。
Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, the reaction incompletion and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with the commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagents, however, were dramatically different with iodoacetamide giving the highest number of peptides with alkylated cysteine and the lowest number of peptides with incomplete cysteine alkylation and side reactions. Alkylation conditions with iodoacetamide were further optimized. To identify more peptides with cysteine, Thiopropyl-Sepharose 6B resins were used to enrich them, and the optimal conditions were employed for the reduction and alkylation. The enrichment resulted in over three times more cysteine-containing peptides than without enrichment. Systematic evaluation of the reduction and alkylation with different reagents can aid in a better design of bottom-up proteomic experiments.
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影响因子：
64.5
作者：
Huttlin EL;Jedrychowski MP;Elias JE;Goswami T;Rad R;Beausoleil SA;Villén J;Haas W;Sowa ME;Gygi SP
通讯作者：
Gygi SP
DOI：
10.1016/j.bbrc.2014.02.041
发表时间：
2014-03-21
影响因子：
3.1
作者：
Catherman, Adam D.;Skinner, Owen S.;Kelleher, Neil L.
通讯作者：
Kelleher, Neil L.
影响因子：
8.4
作者：
Chen W;Smeekens JM;Wu R
通讯作者：
Wu R
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
3.3
作者：
Cottrell, John S.
通讯作者：
Cottrell, John S.