Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips
Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips
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DOI：
10.1038/nprot.2007.261
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Ishihama, Yasushi
中科院分区：
文献类型：
--
作者：
Rappsilber, Juri;Mann, Matthias;Ishihama, Yasushi
基于质谱（MS）的蛋白质组学测量通过蛋白水解切割从蛋白质衍生的肽。在通过基质辅助激光解吸/电离-串联质谱（MALDI-MS/MS）、纳米电喷雾-MS/MS（NanoES-MS/MS）或液相色谱-MS/MS（LC-MS/MS）进行分析之前，肽混合物需要进行清洁、浓缩，并且通常需要选择性富集或预分馏，为此，我们采用简单、自制且非常经济的停止和去提取尖端（StageTips）。StageTips是一种普通的移液器吸头，含有由嵌入聚四氟乙烯网中的具有反相、阳离子交换或阴离子交换表面的珠粒制成的非常小的圆盘。珠粒的固定性质允许具有不同表面的圆盘的灵活组合以获得多功能尖端。可以将含有不同表面官能度的微球和用于磷酸肽富集的松散珠粒如二氧化钛和氧化锆组合。还演示了如何将其纳入自动化工作流程。脱盐和浓缩大约需要5分钟，而分馏或富集大约需要30分钟。
Mass spectrometry (MS)-based proteomics measures peptides derived from proteins by proteolytic cleavage. Before performing the analysis by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS), nanoelectrospray-MS/MS (NanoES-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS), the peptide mixtures need to be cleaned, concentrated and often selectively enriched or pre-fractionated, for which we employ simple, self-made and extremely economical stop-and-go-extraction tips (StageTips). StageTips are ordinary pipette tips containing very small disks made of beads with reversed phase, cation-exchange or anion-exchange surfaces embedded in a Teflon mesh. The fixed nature of the beads allows flexible combination of disks with different surfaces to obtain multi-functional tips. Disks containing different surface functionalities and loose beads such as titania and zirconia for phosphopeptide enrichment can be combined. Incorporation into an automated workflow has also been demonstrated. Desalting and concentration takes approximately 5 min while fractionation or enrichment takes approximately 30 min.