Collision Induced Unfolding Classifies Ligands Bound to the Integral Membrane Translocator Protein.

Membrane proteins represent most current therapeutic targets, yet remain understudied due to their insolubility in aqueous solvents and generally low yields during purification and expression. Ion mobility-mass spectrometry and collision induced unfolding experiments have recently garnered attention as methods capable of directly detecting and quantifying ligand binding within a wide range of membrane protein systems. Despite prior success, ionized surfactant often creates chemical noise patterns resulting in significant challenges surrounding the study of small membrane protein-ligand complexes.

Hypoxia Inducible Factors Modulate Mitochondrial Oxygen Consumption and Transcriptional Regulation of Nuclear-Encoded Electron Transport Chain Genes.

Hypoxia inducible factor-1 (HIF1) is a stress-responsive nuclear transcription factor that is activated with a decrease in oxygen availability. HIF1 regulates the expression of genes involved in a cell's adaptation to hypoxic stress, including those with mitochondrial specific function. To gain a more comprehensive understanding of the role of HIF1 in mitochondrial homeostasis, we studied the link between hypoxia, HIF1 transactivation, and electron transport chain (ETC) function.

Evaluation of ion activation strategies and mechanisms for the gas-phase fragmentation of sulfoquinovosyldiacylglycerol lipids from Rhodobacter sphaeroides.

Sulfoquinovosyldiacylglycerol (SQDG) lipids, found in plants and photosynthetic bacteria, can substitute for phospholipids under phosphate limiting conditions. Here, various low-energy ion activation strategies have been evaluated for the identification and characterization of deprotonated SQDG lipids from a crude membrane lipid extract of Rhodobacter sphaeroides, using collision- induced dissociation - tandem mass spectrometry (CID-MS/MS) in either a triple quadrupole mass spectrometer or in a hybrid quadrupole ion trap-multipole mass spectrometer coupled with high resolution / accurate mass analysis capabilities. In the triple quadrupole instrument, using energy resolved CID-MS/MS experiments, the SQDG head group specific product ion at m/z 225 (C(6)H(9)O(7)S(-)), rather than m/z 81 (SO(3)H(-)), was determined to provide the greatest sensitivity for SQDG lipid detection, and is therefore the preferred `fingerprint' ion for the identification of this lipid class from within complex lipid mixtures when using precursor ion scan mode MS/MS experiments.

Combined genetic and metabolic manipulation of lipids in Rhodobacter sphaeroides reveals non-phospholipid substitutions in fully active cytochrome c oxidase.

A specific requirement for lipids, particularly cardiolipin (CL), in cytochrome c oxidase (CcO) has been reported in many previous studies using mainly in vitro lipid removal approaches in mammalian systems. Our accompanying paper shows that CcO produced in markedly CL-depleted Rhodobacter sphaeroides displays wild-type properties in all respects, likely allowed by quantitative substitution with other negatively charged lipids. To further examine the structural basis for the lipid requirements of R.

Characterization of ornithine and glutamine lipids extracted from cell membranes of Rhodobacter sphaeroides.

The identification and structural characterization of a series of ornithine lipids extracted from the cell membranes of wild-type Rhodobacter sphaeroides, as well as from a glycerophosphocholine-deficient strain, have been achieved by multistage tandem mass spectrometry of their protonated and deprotonated precursor ions in a linear quadrupole ion trap. Systematic examination of the multistage gas-phase fragmentation reactions of these ions, combined with the use of hydrogen/deuterium exchange, has enabled the pathways responsible for sequential losses of the 3-hydroxy linked fatty acyl chain and the amide linked 3-OH fatty acyl chain from these lipids, as well as for formation of the previously reported ornithine specific positively charged "fingerprint" ion at m/z 115, to be determined. Additionally, the fragmentation pathways responsible for formation of a previously unreported ornithine lipid head group-specific product ion at m/z 131 in negative ion mode have been examined.