Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification
Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification
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DOI：
10.1021/ac9911600
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发表时间：
2000-03-15
影响因子：
7.4
通讯作者：
Bradbury, EM
中科院分区：
文献类型：
--
作者：
Chen, X;Smith, LM;Bradbury, EM
质谱仪测定的蛋白质水解肽的质量图谱为蛋白质的鉴定提供了一种主要的方法。蛋白质通常通过蛋白质水解肽的测量和计算的m/z值之间的最佳匹配来识别。然而，唯一的鉴定在很大程度上依赖于多肽电离产生的碎片离子的质量准确性和序列覆盖率，在没有超高仪器精度的情况下，有可能通过将富含稳定同位素的选定氨基酸残基(S)加入到蛋白质序列中来增加特定蛋白水解肽指定的特异性。在这里，我们报告了这种产生残基特异性质量标记蛋白水解肽的新方法，用于准确和高效的蛋白质鉴定。选定的氨基酸被标记为C-13/N-15/H-2，并在细胞培养过程中以特定序列的方式并入蛋白质中。这些标记的氨基酸中的每一个都带有以其单同位素分布模式编码的定义的质量变化。通过它们的特征模式，带有质量标签的多肽可以很容易地在质谱图中与其他多肽区分开来。这种方法。识别独特蛋白质的能力也可以扩展到蛋白质复合体，并将显著提高蛋白质识别的数据搜索特异性、效率和准确性。
Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization, Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with C-13/N-15/H-2 and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method. of identifying unique proteins can also be;extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.