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Sampling of Tissues with Laser Ablation for Proteomics: Comparison of Picosecond Infrared Laser and Microsecond Infrared Laser.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2019-01-29 , DOI: 10.1021/acs.jproteome.9b00009
Andrey Krutilin 1 , Stephanie Maier 1 , Raphael Schuster 2 , Sebastian Kruber 1 , Marcel Kwiatkowski 3 , Wesley D Robertson 1 , Nils-Owe Hansen 1 , R J Dwayne Miller 1, 4 , Hartmut Schlüter 5
Affiliation  

It was recently shown that sampling of tissues with a picosecond infrared laser (PIRL) for analysis with bottom-up proteomics is advantageous compared to mechanical homogenization. Because the cold ablation of tissues with PIRL irradiation is soft, proteins remain intact and even enzymatic activities are detectable in PIRL homogenates. In contrast, it was observed that irradiation of tissues with a microsecond infrared laser (MIRL) heats the tissue, thereby causing significant damage. In this study, we investigated the question if sampling of tissues with a MIRL for analysis of their proteomes via bottom-up proteomics is possible and how the results are different from sampling of tissues with a PIRL. Comparison of the proteomes of the MIRL and PIRL tissue homogenates showed that the yield of proteins identified by bottom-up proteomics was larger in PIRL homogenates of liver tissue, whereas the yield was higher in MIRL homogenates of muscle tissue, which has a significantly higher content of connective tissue than liver tissue. In the PIRL homogenate of renal tissue, enzymatic activities were detectable, whereas in the corresponding MIRL homogenate, enzymatic activities were absent. In conclusion, MIRL and PIRL pulses are suited for sampling tissues for bottom-up proteomics. If it is important for bottom-up proteomic investigations to inactivate enzymatic activities already in the tissue before its ablation, MIRL tissue sampling is an option, because the proteins in the tissues are denatured and inactivated by the heating of the tissue during irradiation with MIRL irradiation prior to the ablation of the tissue. This heating effect is absent during irradiation of tissue with a PIRL; therefore, sampling of tissues with a PIRL is a choice for purifying enzymes, because their activities are maintained.

中文翻译:

用激光消融对蛋白质组学进行组织采样:皮秒红外激光和微秒红外激光的比较。

最近显示,与机械均质化相比,使用皮秒红外激光(PIRL)进行组织采样以进行自下而上的蛋白质组学分析是有利的。由于用PIRL辐照进行的组织冷消融很软,因此蛋白质仍保持完整,甚至在PIRL匀浆中也可检测到酶活性。相反,观察到用微秒红外激光(MIRL)照射组织会加热组织,从而造成明显的损伤。在这项研究中,我们调查了以下问题:使用MIRL进行组织采样以通过自下而上的蛋白质组学分析其蛋白质组是否可能,其结果与使用PIRL进行组织采样有何不同。MIRL和PIRL组织匀浆的蛋白质组的比较表明,由下而上的蛋白质组学鉴定的蛋白质的产量在肝组织的PIRL匀浆中较高,而在肌肉组织的MIRL匀浆中的产量较高,后者的含量明显更高结缔组织比肝组织好。在肾组织的PIRL匀浆中,可检测到酶活性,而在相应的MIRL匀浆中,没有酶活性。总之,MIRL和PIRL脉冲适用于从下至上蛋白质组学的组织采样。如果对于自下而上的蛋白质组学研究重要的是在消融之前使组织中已经存在的酶活性失活,则可以选择MIRL组织取样,因为在消融组织之前,在用MIRL辐射进行辐照的过程中,组织的蛋白质因组织的加热而变性和失活。在用PIRL照射组织的过程中不存在这种加热效果。因此,使用PIRL对组织进行采样是纯化酶的一种选择,因为可以保持酶的活性。
更新日期:2019-02-07
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