The effect of elevating the pressure in the interface region of an electrospray ionization orthogonal time-of-flight mass spectrometer on the ion intensity of different noncovalent protein assemblies has been investigated. Elevating the pressure in the interface region generally led to an enhanced detection of high m/z ions. The optimum pressure was found to be dependent on the m/z value of the ions. This pressure effect should be carefully addressed when relating ion abundance in the mass spectra to solution phase abundance of noncovalent protein assemblies.

Human striated muscle samples, from male control and Duchenne muscular dystrophy-affected children, were subjected to cluster-time-of-flight secondary ion mass spectrometry (cluster-ToF-SIMS) imaging using a 25 keV Bi 3 1 liquid metal ion gun under static SIMS conditions. Spectra and ion density maps, or secondary ion images, were acquired in both positive and negative ion mode over several areas of 500 3 500 mm 2 (image resolution, 256 3 256 pixels). Characteristic distributions of various lipids were observed. Vitamin E and phosphatidylinositols were found to concentrate within the cells, whereas intact phosphocholines accumulated over the most damaged areas of the dystrophic muscles, together with cholesterol and sphingomyelin species. Fatty acyl chain composition varied depending on the region, allowing estimation of the local damage extent.-Tahallah, N., A. Brunelle, S. De La Porte, and O. Laprévote. Lipid mapping in human dystrophic muscle by cluster-time-of-flight secondary ion mass spectrometry imaging. J. Lipid Res. 2008. 49: 438-454.

FprA is a mycobacterial oxidoreductase that catalyzes the transfer of reducing equivalents from NADPH to a protein acceptor. We determined the atomic resolution structure of FprA in the oxidized (1.05 A resolution) and NADPH-reduced (1.25 A resolution) forms. The comparison of these FprA structures with that of bovine adrenodoxin reductase showed no significant overall differences. Hence, these enzymes, which belong to the structural family of the disulfide oxidoreductases, are structurally conserved in very distant organisms such as mycobacteria and mammals. Despite the conservation of the overall fold, the details of the active site of FprA show some peculiar features. In the oxidized enzyme complex, the bound NADP+ exhibits a covalent modification, which has been identified as an oxygen atom linked through a carbonylic bond to the reactive C4 atom of the nicotinamide ring. Mass spectrometry has confirmed this assignment. This NADP+ derivative is likely to form by oxidation of the NADP+ adduct resulting from nucleophilic attack by an active-site water molecule. A Glu-His pair is well positioned to activate the attacking water through a mechanism analogous to that of the catalytic triad in serine proteases. The NADP+ nicotinamide ring exhibits the unusual cis conformation, which may favor derivative formation. The physiological significance of this reaction is presently unknown. However, it could assist with drug-design studies in that the modified NADP+ could serve as a lead compound for the development of specific inhibitors.