Single-nucleotide polymorphisms and haplotype analysis in beta-defensin genes in different ethnic populations.

Beta-defensins are cationic antimicrobial peptides expressed by epithelial cells and exhibit antibacterial, antifungal, and antiviral properties. The defensins are part of the innate host defense network and may have a significant protective role in the oral cavity and other mucosa. Defects or alteration in expression of the beta-defensins may be associated with susceptibility to infection and mucosal disorders. We examined the occurrence of single-nucleotide polymorphisms (SNPs) in the human beta-defensin genes DEFB1 and DEFB2 encoding human beta-defensin-1 and -2 (hBD-1, hBD-2), respectively, in five ethnic populations and defined haplotypes in these populations. Fifteen SNPs were identified in both DEFB1 and DEFB2. Coding region SNPs were found in very low frequency in both genes. One nonsynonymous DEFB1 SNP, G1654A (Val --> Ile), and one nonsynonymous DEFB2 SNP, T2312A (Leu --> His), were identified. Seven sites in each gene exhibited statistically significant differences in frequency between ethnic groups, with the greatest variation in the promoter and in the 5'-untranslated region of DEFB1. DEFB1 displayed 10 common haplotypes, including one cosmopolitan haplotype. Eight common haplotypes were found in DEFB2, including one cosmopolitan haplotype shared among all five ethnic groups. Our results show that genotypic variability among ethnic groups will need to be addressed when performing associative genetic studies of innate defense mechanisms and susceptibility to disease.

Loss of charged versus neutral heme from gaseous holomyoglobin ions.

The dissociation of holomyoglobin ions ranging in charge state from +10 to +2 has been studied using collisional activation in a quadrupole ion trap. Collisional activation times and amplitudes were varied to investigate the effects of these variables on dissociation of the heme group from the holoprotein. The onset of neutral heme loss occurs at a lower activation amplitude than loss of charged heme. For solutions of ferri-myoglobin, charged heme loss was prominent for +10 to +4 holomyoglobin ions, while neutral heme loss product was found to be dominant for charge states +3 and +2. For any given charge state, activation of holomyoglobin ions from a solution containing primarily ferro-myoglobin yielded significantly more abundant neutral heme loss products than was observed for activation of ions from solutions containing primarily ferri-myoglobin. The relative concentrations of the two oxidation states were shown to be affected by redox chemistry within the nano-electrospray emitter used in this work. Results from a double activation experiment revealed that the precursor ions of a given charge state contained a mixture of two populations, with ferro-myoglobin giving rise to neutral heme loss upon dissociation and ferri-myoglobin yielding charged heme. No evidence for electron transfer upon collisional activation of ferri-myoglobin ions was observed. Furthermore, little or no evidence for electron transfer associated with ion/ion reactions with anions derived from perfluoro-1,3-dimethylcyclohexane was observed. Definitive results could not be drawn for the lowest precursor ion charge states (+3 and +2) due to low dissociation efficiencies.

Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry.

One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.