Using Top-Down Mass Spectrometry to Identify and Characterize Endogenous Membrane Protein Complexes

Abstract

Given the key role of integral membrane proteins as transporters, channels, and signal transducers, it is necessary to understand their function and characteristics. Interestingly, approximately 30% of all proteins are membrane proteins, but there are very few fully characterized membrane proteins. This disparity is due to the practical problems of working with membrane proteins, specifically difficulties with expressing, solubilizing, and purifying. Thus, we have designed a simplified way to express membrane proteins using E coli. and purify them using ion exchange column chromatography followed by size exclusion chromatography. This purification technique results in concentrated fractions containing a maximum of four membrane proteins each which allows for easier analysis. The analysis of these endogenous proteins was performed using native top down mass spectrometry in hopes of identifying the proteins in the mixture and to identify their post translational modifications. I was able to find the mass of certain membrane proteins, but I was unable to analyze the MS2 spectra from these proteins. This is due to the fact that native top down mass spectrometry of larger membrane proteins can produce complex spectra that become difficult to analyze and match with a database given the current available software. This study gives an overview on the complexities of collecting and analyzing data from a mixture of unknown membrane proteins.