We describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one- and two-dimensional polyacrylamide gel electrophoresis. The approach is based on the use of an isotopically labeled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. The method allows quantitation of the changes occurring in spots or bands that contain more than one protein and has a greater dynamic range than most staining methods. Since the reagent carries a fixed positive charge under acidic conditions and labels only the N-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. The sequences can easily be extracted for homology searches instead of using indirect mass spectral-based searches and are independent of posttranslational modifications.
我们描述了一种比较定量和从头肽测序的方法,用于通过标准色谱法或一维和二维聚
丙烯酰胺凝胶电泳分离的蛋白质。该方法基于使用同位素标记试剂(通过质谱法)定量在不同条件下表达的蛋白质消化产物中肽的比例。该方法可以定量包含多种蛋白质的斑点或条带中发生的变化,并且比大多数染色方法具有更大的动态范围。由于试剂在酸性条件下带有固定的正电荷,并且仅标记肽的N端,因此大大简化了串联质谱的解释,从而获得序列信息。可以轻松提取序列进行同源性搜索,而不必使用基于质谱的间接搜索,并且与翻译后修饰无关。