期刊
ANALYTICAL CHEMISTRY
卷 91, 期 12, 页码 7578-7585出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.8b05889
关键词
-
资金
- National Institutes of Health (NIH) National Institute of General Medical Sciences [5P41 GM103391-07]
- NIH National Institute of Allergy and Infectious Diseases [1R01 AI138581-01A1, R01AI132560, K08AI113107]
- NIH Shared Instrumentation Grant Program [1S10OD012359-01]
- Burroughs Wellcome Fund Career Award for Medical Scientists
The ability to target discrete features within tissue using liquid surface extractions enables the identification of proteins while maintaining the spatial integrity of the sample. Here, we present a liquid extraction surface analysis (LESA) workflow, termed microLESA, that allows proteomic profiling from discrete tissue features of similar to 110 mu m in diameter by integrating nondestructive autofluorescence microscopy and spatially targeted liquid droplet micro-digestion. Autofluorescence microscopy provides the visualization of tissue foci without the need for chemical stains or the use of serial tissue sections. Tryptic peptides are generated from tissue foci by applying small volume droplets (similar to 250 pL) of enzyme onto the surface prior to LESA. The microLESA workflow reduced the diameter of the sampled area almost 5-fold compared to previous LESA approaches. Experimental parameters, such as tissue thickness, trypsin concentration, and enzyme incubation duration, were tested to maximize proteomics analysis. The microLESA workflow was applied to the study of fluorescently labeled Staphylococcus aureus infected murine kidney to identify unique proteins related to host defense and bacterial pathogenesis. Proteins related to nutritional immunity and host immune response were identified by performing microLESA at the infectious foci and surrounding abscess. These identifications were then used to annotate specific proteins observed in infected kidney tissue by MALDI FT-ICR IMS through accurate mass matching.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据