Role of accurate mass measurement (±10 ppm) in protein identification strategies employing MS or MS MS and database searching
Role of accurate mass measurement (±10 ppm) in protein identification strategies employing MS or MS MS and database searching
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DOI：
10.1021/ac9810516
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发表时间：
1999-07-15
影响因子：
7.4
通讯作者：
Burlingame, AL
中科院分区：
文献类型：
--
作者：
Clauser, KR;Baker, P;Burlingame, AL
我们描述了质量测量精度的进步，+/-10 ppm（内部校准），对蛋白质鉴定实验的影响。这种能力是通过延迟提取技术与基质辅助激光解吸电离（MALDI）结合使用的反射飞行时间（TOF）质谱仪。这项工作探讨了使用准确的质量测量（从而限制蛋白质消化物中组分的可能元素组成）在搜索蛋白质，基因和EST数据库的策略中的优势，这些数据库采用（a）单独的质量值，（B）来自MS/MS光谱的碎片离子标记，和（c）MS/MS光谱的从头解释。已经发现仅使用分子量值（即，测量的质量）>10个肽质量。当MALDI-TOF仪器能够达到区分肽元素组成所需的+/-0.5-5 ppm质量准确度时，可以将具有>70%序列同一性的同源蛋白质与被分析的蛋白质匹配。单个胰蛋白酶肽的+/-10 ppm测量的母体质量和来自通过MS/MS产生的亚铵离子的接近完整的氨基酸（AA）组成信息的组合能够在数据库中标记肽，因为在蛋白质组中只能发现AA组成的长度>11个AA的少数序列排列，肽MS/MS谱的从头解析可以通过改变我们的MS-Tag程序来完成，以仅计算可能来自准确母体质量和亚铵离子限制的AA组成的序列排列来替换整个数据库。采用一种混合策略，使用从头MS/MS解释，然后进行基于文本的序列相似性搜索的数据库。
We describe the impact of advances in mass measurement accuracy, +/-10 ppm (internally calibrated), on protein identification experiments. This capability was brought about by delayed extraction techniques used in conjunction with matrix-assisted laser desorption ionization (MALDI) on a reflectron time-of-flight (TOF) mass spectrometer. This work explores the advantage of using accurate mass measurement (and thus constraint on the possible elemental composition of components in a protein digest) in strategies for searching protein, gene, and EST databases that employ (a) mass values alone, (b) fragment-ion tagging derived from MS/MS spectra, and (c) de novo interpretation of MS/MS spectra. Significant improvement in the discriminating power of database searches has been found using only molecular weight values (i.e., measured mass) of >10 peptide masses. When MALDI-TOF instruments are able to achieve the +/-0.5-5 ppm mass accuracy necessary to distinguish peptide elemental compositions, it is possible to match homologous proteins having >70% sequence identity to the protein being analyzed. The combination of a +/-10 ppm measured parent mass of a single tryptic peptide and the near-complete amino acid (AA) composition information from immonium ions generated by MS/MS is capable of tagging a peptide in a database because only a few sequence permutations >11 AA's in length for an AA composition can ever be found in a proteome, Dc novo interpretation of peptide MS/MS spectra may be accomplished by altering our MS-Tag program to replace an entire database with calculation of only the sequence permutations possible from the accurate parent mass and immonium ion limited AA compositions. A hybrid strategy is employed using de novo MS/MS interpretation followed by text-based sequence similarity searching of a database.