小分子表面活性剂在胶内酶切增效中的应用

本研究成功地将一种有机小分子表面活性剂RapiGest SF(Waters)用于改进电泳分离的蛋白质的鉴定效率。通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)的肽质量指纹谱,考察了酶切时间、加入量、加样次序、点靶方法对方法灵敏度、蛋白质鉴定率的影响。RapiGest SF浓度为0.5%~1%,在酶切之前加入可获得更多的肽段峰和更高的鉴定率。本方法考染体系的总灵敏度为332fmol,银染体系为664fmol。比较了RapiGest SF与MALDI-TOF-MS和电喷雾质谱(ESI-MS)兼容性,未观察到明显的负影响。方法操作简便,重复性较好,适合鉴定电泳分离的低丰度蛋白质。

Application of Small Molecular Anionic Surfactant on the Efficiency Enhancement of In-gel Digestion

For gel-based proteomics, peptide recovery of in-gel digestion is crucial for mass spectrum quality and protein identification. An anionic surfactant (RapiGest SF,Waters) was successfully used to improve gel-based protein identification. Based on MALDI-TOF-MS peptide mass fingerprinting (PMF), digestion time, the effect of RapiGest SF amount, addition procedure, MALDI-TOF-MS spotting with AnchorChip target (Bruker) were examined on the sensitivity and protein identification ratio, RapiGest SF concentration of 0.5%-1% and addition before enzyme (trypsin) could get more peaks and high protein identification. The total sensitivity of this system was 332 fmol for coomassie staining gel and 664 fmol for silver staining gel respectively. The compatibility of RapiGest SF with MALDI-TOF-MS and ESI mass spectrometry were compared, no apparent negative impact on ESI system was observed. This method was easily carried out with a good reproducibility, which was useful for low abundant protein identification.