Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics
Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics
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DOI：
10.1021/ac0483062
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发表时间：
2005-05-15
影响因子：
7.4
通讯作者：
Smith, RD
中科院分区：
文献类型：
--
作者：
Shen, YF;Zhang, R;Smith, RD
基于反相液相色谱（RPLC）的蛋白质组学分析被广泛实践;然而，提供尖端RPLC性能的变化通常没有被采用，即使它们的益处已经被充分确立。在这里，我们描述了一个自动化的格式20千磅/平方英寸的RPLC系统的蛋白质组学和代谢组学，包括在线耦合的微固相萃取样品加载，并允许电喷雾电离发射器可以很容易地更换。该系统使用50 μ m i. d. x 40-200 cm的熔融石英毛细管，填充有1.4-3 μ m的多孔C18键合的二氧化硅颗粒，以获得1000-1500的复杂肽和代谢物混合物的色谱峰容量。这种分离质量提供了从> 2000种不同的希瓦氏菌蛋白质中鉴定> 12000种不同胰蛋白酶肽的高置信度鉴定（类似于对S. oneidensis蛋白质组）在单个12-h离子阱串联质谱（MS/MS）分析中。重复实验之间的蛋白质鉴定重现性接近90%。平均蛋白质MS/MS鉴定速率超过10个蛋白质/min，在120 min内鉴定了1207个蛋白质，通过5944个不同的肽段进行了归属。基于对人血浆样本的分析，20 kpsi RPLC离子阱MS/MS的蛋白质组学分析动态范围约为10（6），在单次12小时运行中以高置信度鉴定了835种不同的蛋白质。单次运行20 kpsi的HPLC-精确质量MS从代谢组学样品中检测到> 5000种不同的化合物。
Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however, variations providing cutting-edge RPLC performance have generally not been adopted even though their benefits are well established. Here, we describe an automated format 20 kpsi RPLC system for proteomics and metabolomics that includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionization emitters to be readily replaced. The system uses 50 mu m i.d. x 40-200 cm fused-silica capillaries packed with 1.4-3-mu m porous C18-bonded silica particles to obtain chromatographic peak capacities of 1000-1500 for complex peptide and metabolite mixtures. This separation quality provided high-confidence identifications of > 12 000 different tryptic peptides from > 2000 distinct Shewanella oneidensis proteins (similar to 40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The protein identification reproducibility approached 90% between replicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207 proteins were identified in 120 min through assignment of 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS was approximately 10(6) based on analyses of a human blood plasma sample, for which 835 distinct proteins were identified with high confidence in a single 12-h run. A single run of the 20 kpsi RPLC-accurate mass MS detected > 5000 different compounds from a metabolomics sample.