This unit addresses several critical challenges associated with membrane protein crystallography by screening membrane proteins from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebral tissue for biochemical properties favorable for crystallization. First, a tissue sample or cell pellet is obtained. The cells are isolated, washed, and then lysed either by sonication, bead beating, or manual homogenization. Membrane proteins are fractionated from the lysates by centrifugation and solubilized in a mild detergent suitable for crystallization, such as n-dodecyl-β-maltoside (DDM). Detergent extracts are then centrifuged, heat precipitated, and filtered to remove insoluble, thermally unstable, and/or aggregated proteins. Samples are then prepared for analysis by mass spectrometry: proteins are precipitated by methanol/chloroform extraction and subjected to reduction, alkylation, and protease digestion. The resulting peptides are passed through a detergent removal column, desalted, rehydrated in 0.1% formic acid (v/v), and identified by LC-MS/MS. © 2017 by John Wiley & Sons, Inc.
Keywords: crystallography; mass spectrometry; membrane proteins; protein crystallization; proteomics.
Copyright © 2017 John Wiley & Sons, Inc.