News
Ca 2 + /peptide binding in the gas phase Calcium-binding sites in proteins vary in their affinity for Ca2+, depending on their amino-acid sequences. Aspartic acid, glutamic acid, and asparagine, as well as other amino acids that contain carbonyl, carboxyl, or nonaromatic oxygen groups, are generally involved in calcium-binding sites. Previous studies in solution have shown that the affinity for Ca2+ in proteins is also influenced by solvent effects. Michael L. Gross and Olga V. Nemirovskiy of Washington University have investigated calcium/peptide complexes of the form [M - H + Ca]+ and [M + Ca]2+ +i the gas shasee which c p r y p Jiff*
models of a calciumbinding site in a rabbit protein By comparing properties in the gas phase with those in solution the influence of solvent effects can be better understood Calcium/peptide complexes are generated by fast atom bombardment (FAB) and electrospray ionization (ESI) and intro-
duced into a mass spectrometer. Under ESI conditions, both the mono- and divalent ions are produced; however, only the monovalent ion is produced by FAB. The abundances of the ions relative to [M + H]+ and [M + 2H]2+ depend on pH, solvent composition, amino-acid sequence, and type. The formation of [M - H + Ca]+ +i favored under basic conditions and that of [M + Ca]2+ under neutral conditions; however, protonated peptide species are favored under acidic conditions and the competitive binding to Ca2+ is diminished. Conditions optimized to produce abundance of the Ca2+/peptide ions Fragments produced by low energy (LE) collisionally activated decomposition (CAD) of the ESI-produced complexes gave clues to the location of the Ca2+ binding site. LE CAD spectra indicate that binding of Ca2+ to the peptide occurs at deprotonated acid side chains and carbonyl oxygens. High-energy CAD spectra obtained for the FAB-produced complexes show that
Blame it on the gold The studies found that the thiols did indeed oxidize in the dark, but that the Alkanethiols are often used to prepare rate of oxidation varied from complete self-assembled monolayers on various conversion in a few days to no changes substrates; however, the thiols have over two weeks. The different rates were been shown to oxidize in air to alkyl found to be related to the surface morsulfinates and sulfonates. The oxidation phology. As the was initially densities of grain thought to inboundaries involve photocreased and the chemical proamounts of cesses, but the Au(lll)onthe reaction has resurface decently been creased the rate shown to proof oxidization ceed in the dark. increased. The So what is researchers behappening? lieve that the Michael S. grain boundaries Freund, Gregory correspond to S. Ferguson, and defects that give their colleagues at rise to greater Lehigh University reactivitv Morestudied the oxida- STM images of thermally evaporated gold films on glass (a and c) and on silicon (b and tion of alkanethiol d) which show different grain sizes. on some monolayers on surfaces the regold using X-ray photoelectron spectrosore AiidOOl which copy. The gold was thermally evaporated does not bind alkanethink as stronclv as onto glass or silicon supports and the grain Air (11l) O idi d thi l rtorie size was quantitatively assessed by scansorb from the surface under ambient conning tunneling microscopy (STM) 768 A
Analytical Chemistry News & Features, December 1, 1998
the number of populated sites increases with increasing energy. Interactions of Mg2* and Ba2+ with the peptides are also investigated, and the types of fragments produced by LE CAD are found to be similar to those of Ca2+. The abundances of the fragments, however, depend on the sizes of the metal ions. (J. Am. .oc. Mass Spectrom. 1998, 9,1020-28)
Human spliceosome characterized with MS Multiprotein complexes perform or regulate many cellular processes. One such complex is known as the spliceosome, which removes introns (unexpressed gene sequences) during RNA transcription. This complex has been only partially characterized—until now. Matthias Mann (now at Odense University in Denmark) and colleagues at the European Molecular Biology Laboratory (Germany) and Dundee University (United Kingdom) characterized the human spliceosome complex with the combination of 2-D gel electrophoresis, 113110" electrospray MS, and publicly available expressed sequence tag (EST) databases. Human splicing complexes were assembled on a pre-RNA substrate and purified. Proteins that were stably purified with the pre-RNA substrate were separated by 2-D gel electrophoresis. Sixty-nine protein spots were excised, digested with trypsin, and analyzed by nanoelectrospray MS. Screening against a protein database revealed that 41 of those spots (representing 25 proteins) were the products of previously characterized genes. The remaining spots (only 65 could be identified) were characterized with EST clones. Nineteen previously unidentified human proteins copurified with the splicing complexes. The authors acknowledge mat some splicing factors with a greater affinity for other introns might have been lost during the purification process. However, they point out that the high coverage of known splicing factors suggests that the majority of core spliceosome proteins were included in the analysis. Although EST databases contain a relatively small percentage of human genes and the sequences are short fragments, the authors indicate that when combined with MS these databases are sufficient to characterize large multiprotein complexes. (Natt Genet. .199 20(1) 46-50)
