title: Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry.
abstract: Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation.
authors: The Pasarow Mass Spectrometry Laboratory, NPI-Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
authors: Puneet
authors: P
authors: Souda
authors: Christopher M
authors: CM
authors: Ryan
authors: William A
authors: WA
authors: Cramer
authors: Julian
authors: J
authors: Whitelegge
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D002853:Chromatography, Liquid; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010940:Plant Proteins