Institute of Analytical Chemistry of the Academy of Sciences of the Czech Republic, v.v.i., Veverí 97, 602 00 Brno, Czech Republic.
J Mass Spectrom. 2009 Nov;44(11):1613-21. doi: 10.1002/jms.1675.
Proteomic-based description of varieties of barley (Hordeum vulgare L.) is a very important task especially in the food and brewing industry. This study is focused on major storage proteins in barley--hordeins--as a group of proteins soluble in alcohol/water mixtures that shows up significant changes in proteomic profiles for different varieties of barley. Unusual amino acid composition of hordeins with low numbers of lysine and arginine in combination with high content of proline and glutamine complicates their identification in a common proteomic workflow, because tryptic digestion produces just a few peptides amenable for successful mass spectrometric analysis. To increase the number of cleavage sites, in this work, cysteines in hordeins were chemically modified with 2-bromoethylamine (BEA) for their conversion into aminoethylcysteines. These mimic lysine residues and are recognized by trypsin as potential cleavage sites (if not followed by a proline residue) on the C-terminal side of the modified cysteine. Small extent of side reactions (towards histidine, N-terminus of the peptide, methionine, and also here the newly discovered reaction towards aminoethylcysteine) during modification with BEA could be observed after a longer period of reaction but this did not hinder the analysis when optimal conditions were used. Application of trypsin for in-gel digestion of hordeins, previously modified chemically with BEA, provided a higher number of short peptides and their subsequent mass spectrometric analysis resulted in an improved identification of hordeins. This approach can also be used for the analysis of other similar protein groups (e.g. gliadins in wheat) or other cysteines containing proteins having a low number of lysine and arginine residues in their primary structure.
基于蛋白质组学的大麦（Hordeum vulgare L.）品种描述是一项非常重要的任务，尤其是在食品和酿造行业。本研究集中于大麦中的主要储存蛋白——醇溶蛋白——作为一组在不同大麦品种的蛋白质组学图谱中显示出显著变化的蛋白质。醇溶蛋白的异常氨基酸组成，赖氨酸和精氨酸数量低，脯氨酸和谷氨酰胺含量高，这使得它们在常见的蛋白质组学工作流程中的鉴定变得复杂，因为胰蛋白酶消化只会产生少量适合成功进行质谱分析的肽。为了增加切割位点的数量，在这项工作中，使用 2-溴乙胺（BEA）对醇溶蛋白中的半胱氨酸进行化学修饰，将其转化为氨基乙基半胱氨酸。这些模拟赖氨酸残基，被胰蛋白酶识别为潜在的切割位点（如果不被脯氨酸残基紧随其后），位于修饰半胱氨酸的 C 末端。在 BEA 修饰过程中，可以观察到较小程度的副反应（朝向组氨酸、肽的 N 末端、蛋氨酸，以及此处新发现的朝向氨基乙基半胱氨酸的反应），但在使用最佳条件时，这并不会妨碍分析。BEA 化学修饰后，使用胰蛋白酶对醇溶蛋白进行胶内消化，提供了更多的短肽，随后对这些肽进行质谱分析，提高了醇溶蛋白的鉴定。这种方法也可用于分析其他类似的蛋白质组（例如小麦中的麦谷蛋白）或其他在其一级结构中赖氨酸和精氨酸数量低的含半胱氨酸的蛋白质。
