Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics
Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics
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DOI：
10.1021/ac026117i
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发表时间：
2003-02-01
影响因子：
7.4
通讯作者：
Mann, M
中科院分区：
文献类型：
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作者：
Rappsilber, J;Ishihama, Y;Mann, M
蛋白质组学在很大程度上依赖于最佳的样品制备。特别是，蛋白质消化和质谱分析之间的接口对分析的整体质量和灵敏度有很大影响。我们在这里描述了一种新的程序，其中嵌入在聚四氟乙烯网珠的一个非常小的磁盘被放置为微柱到移液器提示。对于Slop And Go萃取，该程序已使用市售材料（C18 Empore硅藻土（3M，Minneapolis，MN））作为玻璃料和分离材料来实施。该磁盘是在一个简单而快速的过程中产生一个方便和完全可靠的程序，用于生产自包装的微柱在移液器吸头。仅通过变窄的吸头将其固定在无障碍物的位置，确保分析物的最佳加载和洗脱。在1分钟内方便地放置5个盘，对于填充柱使用手动力以300 μ L/分钟的速度添加），同时消除了堵塞的可能性。C（18）-StageTips（柱床：直径0.4 mm，长度0.5 mm）的装载容量为2 - 4 μ g蛋白质消化物，可通过使用更大直径或堆叠的盘来增加。可定量回收5飞摩尔的胰蛋白酶BSA消化物。我们发现Stage系统非常适合作为蛋白质组学的通用样品制备系统。
Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for Slop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 muL/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C(18)-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 mug of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.