Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching
Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching
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DOI：
10.1021/ac9913210
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发表时间：
2000-03-15
影响因子：
7.4
通讯作者：
Aebersold, R
中科院分区：
文献类型：
--
作者：
Goodlett, DR;Bruce, JE;Aebersold, R
本文描述了一种利用单个肽的精确质量和特定序列约束，通过序列数据库搜索快速、明确地鉴定蛋白质的方法。使用电喷雾电离-傅里叶变换离子回旋共振质谱法测量肽质量，精度为1ppm。在肽序列中是否存在半胱氨酸残基被用作数据库搜索约束，以减少潜在的数据库命中数。含有半胱氨酸的多肽在多肽混合物中检测，通过将氯掺入半胱氨酸残基专用的一般烷基化试剂中。二级搜索约束包括用于蛋白质消化的蛋白酶的特异性和凝胶电泳估计的蛋白质分子质量。氯的天然同位素分布以独特的同位素模式编码含半胱氨酸肽，使质谱自动筛选成为可能。使用所有6118个可能的酵母开放阅读框作为数据库，对酵母裂解物中的肽标准蛋白和未知蛋白进行了验证。根据密码子偏倚的计算判断，使用这种新方法可以从酵母裂解物中鉴定出低丰度的蛋白质，而传统的方法，如串联质谱法，通过数据依赖的获取或质量映射，无法从酵母裂解物中鉴定出低丰度的蛋白质。
A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.