Pressure-Assisted Lysis of Mammalian Cell Cultures Prior to Proteomic Analysis

Abstract

Mass spectrometry-based proteomic analysis of specimens derived from mammalian cell cultures and tissues provides informative views into the complex molecular mechanisms of cellular processes. Good control, efficiency, and reproducibility of protein extraction from cells and tissues are essential aspects for downstream proteomic assays, as well as for other diverse biological and basic research applications. However, common cell lysis techniques suffer from multiple limitations including the lack of reproducibility in protein extraction, covalent alteration of the sample molecules, and misrepresentation of subpopulations of cellular proteins, among others. In this work we have examined the effect of altering both the lysis buffer and the mechanism of lysis in order to enhance proteomic coverage from a cell culture sample. Ultra-high hydrostatic pressure cycling and the addition of fluorinated alcohol in the lysis buffer have been tested due to their proposed individual abilities to solubilize and change conformations of proteins in order to facilitate downstream enzymatic digestion and LC-MS/MS-based proteomic analysis. The resulting data shows that both changes in the cell lysis method demonstrate enrichment for different classes of proteins and protein classes, including membrane- and organelle-associated proteins, meanwhile increasing the total number of recovered proteins and peptides when compared to the conventional lysis methods.