Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI Fourier transform ion cyclotron resonance mass spectrometry
Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI Fourier transform ion cyclotron resonance mass spectrometry
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DOI：
10.1021/ac000497v
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发表时间：
2000-09-15
影响因子：
7.4
通讯作者：
Marto, JA
中科院分区：
文献类型：
--
作者：
Martin, SE;Shabanowitz, J;Marto, JA
亚飞摩尔多肽序列分析使用微毛细管高效液相色谱柱，集成的纳米电喷雾发射器，直接连接到傅立叶变换离子回旋共振质谱仪。准确的质量(+/-0.010 Da)肽图是从标准的六种蛋白质消化混合物中产生的，其主要成分跨越了1000：1的浓度动态范围。对OWL数据库中类似189 000个条目的迭代搜索很容易识别出每个蛋白质，具有高序列覆盖率(20-60%)，从柱上加载的低至10amol。此外，一种简单的可变流量高效液相色谱装置提供了400-amol水平的胰蛋白酶多肽的在线串联质谱分析。使用SEQUEST在非冗余蛋白质数据库中搜索类似于28万个条目的MS/MS数据。准确的前体和产物离子质量信息容易识别不同的初级氨基酸序列：天冬酰胺与天冬氨酸(Delta m=0.98Da)和谷氨酰胺与赖氨酸(Delta m=0.036 Da)。
Subfemtomole peptide sequence analysis has been achieved using microcapillary HPLC columns, with integrated nanoelectrospray emitters, coupled directly to a Fourier transform ion cyclotron resonance mass spectrometer. Accurate mass (+/-0.010 Da) peptide maps are generated from a standard six-protein digest mixture, whose principle components span a concentration dynamic range of 1000:1. Iterative searches against similar to 189 000 entries in the OWL database readily identify each protein, with high sequence coverage (20-60%), from as little as 10 amol loaded on-column. In addition, a simple variable-flow HPLC apparatus provides for on-line tandem mass spectrometric analysis of tryptic peptides at the 400-amol level. MS/MS data are searched against similar to 280 000 entries in a nonredundant protein database using SEQUEST. Accurate precursor and product ion mass information readily identifiers primary amino acid sequences differing by:asparagine vs aspartic acid (Delta m = 0.98 Da) and glutamine vs lysine (Delta m = 0.036 Da).