Patel Ekta, Clench Malcolm R, West Andy, Marshall Peter S, Marshall Nathan, Francese Simona
Biomolecular Research Centre, Sheffield Hallam University, Sheffield, S1 1WB, UK.
J Am Soc Mass Spectrom. 2015 Jun;26(6):862-72. doi: 10.1007/s13361-015-1140-z. Epub 2015 Apr 28.
Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual's fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis.
尽管原位蛋白水解策略最近有所改进,但仍需要更高的效率来增加检测到的肽段数量和相关离子强度,从而获得一套完整且可靠的用于诊断或预后目的的生物标志物。在本文介绍的研究中,展示了一项系统研究的提取物,该研究调查了一系列有助于胰蛋白酶蛋白水解活性的表面活性剂。方法开发在指纹上进行了试验;这种样本是由于个体指尖上的汗液在接触时转移到表面而形成的。由于汗液携带大量生物分子,包括肽和蛋白质,指纹有可能成为非侵入性预后或诊断筛查的非常有价值的样本。最近的一项研究表明,使用基质辅助激光解吸电离质谱分析(MALDI MSP)有机会快速检测指纹中的肽和小蛋白质。然而,完整检测的灵敏度较低,且无法进行物种鉴定;因此,采用了一种涉及原位蛋白水解的鸟枪法蛋白质组学方法。数据表明,在指纹中,以更高百分比使用MEGA - 8作为表面活性剂以及不同洗涤剂的组合,可以对现有方法进行进一步改进。此外,通常用于溶液消化的Rapigest SF首次被证明也能成功用于原位蛋白水解。