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采用在线 LC-MS 策略优化半胱氨酸残基的烷化作用:使用卤乙酰胺试剂混合物的益处。

Optimization of cysteine residue alkylation using an on-line LC-MS strategy: Benefits of using a cocktail of haloacetamide reagents.

机构信息

Atlantic Cancer Research Institute, Hotel Dieu Pavilion, 35 Providence Street, Moncton, New Brunswick, E1C8X3, Canada; Department of Process Engineering and Applied Science, Dalhousie University, Sexton Campus Chemical Engineering Building, PO Box 15000, Halifax, Nova Scotia, B3H4R2, Canada.

Atlantic Cancer Research Institute, Hotel Dieu Pavilion, 35 Providence Street, Moncton, New Brunswick, E1C8X3, Canada.

出版信息

Anal Biochem. 2021 Apr 15;619:114137. doi: 10.1016/j.ab.2021.114137. Epub 2021 Feb 11.

DOI:10.1016/j.ab.2021.114137
PMID:33582115
Abstract

Several common reagents for the alkylation of cysteine residues of model intact proteins were evaluated for reaction speed, yield of alkylated product and degree of over-alkylation using an online LC-MS platform. The efficiency of the alkylation reaction is found to be dependent on the (1) reagent, (2) peptide/protein, (3) reagent concentration and (4) reaction time. At high reagent concentrations, iodoacetic acid was found to produce significant levels of over-alkylation products wherein methionine residues become modified. For optimal performance of the alkylation reaction, we found the use of a cocktail of chloroacetamide, bromoacetamide and iodoacetamide worked best. The alkylating efficiency of each haloacetamide is a balance between the characteristics of the halogen leaving group and the steric hindrance of the alkylation site on the peptide or protein. A key aspect of using a cocktail of haloacetamides is that they all produce the same modification (+57.0209 Da) to the cysteine residues of the protein while the alkylation efficiency of each site may differ for each of the three reagents. Over-alkylation effects appear to be lower with the cocktail due to a lower concentration of each reagent. The haloacetamide cocktail could be useful when considering complex mixtures of proteins.

摘要

几种用于修饰模型完整蛋白质半胱氨酸残基的常见试剂,使用在线 LC-MS 平台评估其反应速度、烷基化产物的产率和过度烷基化程度。发现烷基化反应的效率取决于 (1) 试剂、(2) 肽/蛋白质、(3) 试剂浓度和 (4) 反应时间。在高试剂浓度下,碘乙酸被发现会产生大量的过度烷基化产物,其中蛋氨酸残基被修饰。为了获得最佳的烷基化反应性能,我们发现使用氯乙酰胺、溴乙酰胺和碘乙酰胺的混合物效果最佳。每种卤代乙酰胺的烷基化效率是卤原子离去基团的特性和肽或蛋白质上烷基化位点的空间位阻之间的平衡。使用卤代乙酰胺混合物的一个关键方面是,它们都为蛋白质的半胱氨酸残基产生相同的修饰(+57.0209 Da),而每个位点的烷基化效率可能因三种试剂中的每一种而异。由于每种试剂的浓度较低,因此混合物的过度烷基化效应似乎较低。当考虑复杂的蛋白质混合物时,卤代乙酰胺混合物可能很有用。

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