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Sample preparation protocol for proteomic analysis of isolated lysosomes and whole cell extracts
Output Details
Description
Mass spectrometry-based proteomics has emerged as fundamental technique to study functional changes of proteome including post translational modifications. Sample preparation is key for an effective and reproducible identification and quantification for proteomic analysis. Here, we describe a step wise protocol for samples derived from cell lines models or isolated human cells. The protocol has been optimised for organelle pulldown preps. To maximize proteomic coverage, we deploy a strong detergent (2% SDS), as well as high energy sonication to ensure complete solubilization of tissue/cellular proteins. We describe a facile protocol for straightforward capture of solubilized protein samples on a S-trap column that allows removal of SDS and other components that interfere with protease digestion. We provide an optimized trypsin/Lys-C protease digestion protocol to maximize protein digestion.
Identifier (DOI)
10.17504/protocols.io.q26g7p2d8gwz/v1