Sanchez Aniel, Ramos Yassel, Solano Yanni, Gonzalez Luis Javier, Besada Vladimir, Betancourt Lazaro, Gil Jeovanis, Alvarez Felix, Rodriguez Meilyn, Perez Lincidio, Pujol Merardo, Padron Gabriel
Department of Proteomics, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Rapid Commun Mass Spectrom. 2007;21(14):2237-44. doi: 10.1002/rcm.3079.
We report here a method for the identification of free or blocked N-terminal peptide of in-gel digested isolated proteins. The primary amino groups of the gel-entrapped protein are blocked with normal acetic or succinic anhydride, and the protein is digested with a high-specificity protease. The generated peptides are treated with an equimolar mixture of normal and deuterated acetic anhydride. Upon mass spectrometric analysis internal peptides display a complex isotopic ion distribution while the N-terminal peptide shows a normal isotopic ion distribution. The procedure was applied to the identification of the N-terminus of individual and protein mixtures isolated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).
我们在此报告一种用于鉴定凝胶内消化分离蛋白质的游离或封闭 N 端肽的方法。凝胶包埋蛋白质的伯氨基用普通乙酸酐或琥珀酸酐封闭，然后用高特异性蛋白酶消化该蛋白质。生成的肽用普通乙酸酐和氘代乙酸酐的等摩尔混合物处理。质谱分析时，内部肽显示出复杂的同位素离子分布，而 N 端肽显示出正常的同位素离子分布。该方法应用于通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS-PAGE）分离的单个蛋白质和蛋白质混合物 N 端的鉴定。
