Automation of nanoscale microcapillary liquid chromatography-tandem mass spectromentry with a vented column
Automation of nanoscale microcapillary liquid chromatography-tandem mass spectromentry with a vented column
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DOI：
10.1021/ac025529o
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发表时间：
2002-07-01
影响因子：
7.4
通讯作者：
Gygi, SP
中科院分区：
文献类型：
--
作者：
Licklider, LJ;Thoreen, CC;Gygi, SP
为了使纳米级微毛细管液相色谱-串联质谱（LC-MS/MS）分析的样品导入步骤完全自动化，将75 μ w × 14 cm的毛细管柱与商用自动进样器连接，使用一种新颖的程序，允许稀释肽样品以高达5 μ l min（-1）的流速从AS环注射器转移到纳米级柱。通过在5- mom ODS-AQ改性二氧化硅填充床入口后2 cm处构建排气口，演示了在柱上富集和脱盐的大样本量（bbb40 muL）。盐和未保留的溶质通过通风口被去除，这使得柱洗涤独立于床层进入电喷雾源的延续。测试肽混合物的分离显示出50-nL的洗脱峰体积，检测水平低至亚毫摩尔。此外，一种高度复杂的多肽混合物（铜绿假单胞菌的外膜制备）与100多种蛋白质进行了有效的分离，通过单次反相LC-MS/MS分析鉴定。最后，在多维层析/串联质谱实验中，利用通气柱（v柱）进行在线分离，从胰蛋白酶化酵母裂解物中获得大量强阳离子交换层析馏分，在完全自动化的方式下进行脱盐、浓缩和分析。构建和使用v柱的程序需要在使用直径为100微米及更小的柱的纳米lc - ms分析的当前方法和设备中进行最小的更改。
To fully automate the sample introduction step for nano-scale microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, 75 mum W. x 14 cm capillary columns were interfaced with a commercial autosampler instrument using a novel procedure which allowed dilute peptide samples to be transferred from the AS loop injector to the nanoscale column at flow rates up to 5 muL min(-1). On-column enrichment and desalting was demonstrated for large sample volumes (>40 muL) by constructing a vent 2 cm after the entrance to the packed bed of 5-mum ODS-AQ modified silica. Salts and nonretained solutes were removed via the vent, which allowed for column washing independent of the continuation of the bed into the electrospray source. Separations of test peptide mixtures demonstrated 50-nL elution peak volumes with low- to subfemtomole detection levels. In addition, a highly complex peptide mixture (outer membrane preparation from Psuedemonas aeruginosa) was efficiently separated with more than 100 proteins identified from a single reversed-phase LC-MS/MS analysis. Finally, the vented column (V-column) was utilized for online separations in a multidimensional chromatography/tandem MS experiment where large numbers of strong cation exchange chromatography fractions from a trypsinized yeast lysate were desalted, concentrated, and analyzed in a completely automated fashion. The procedures for constructing and using a V-column require minimal changes in current methods and equipment for nano-LC-MS analyses using columns of 100-mum diameter and smaller.