Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.
Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Twodimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures.
Angle resolved time of flight (TOF) measurements of the fragments produced when allene is photolyzed at 193 nm are described. The two primary processes that have been identified from these measurements are the H+C3H3 and the H2+C3H2 channels. The quantum yields for these first steps are 0.89 and 0.11, respectively. Subsequent photolysis of the C3H3 radical produces H2+C3H, C3H2+H, and C2H2+CH, while the C3H2 produces C3+H2, C2H+CH, and C2H2+C. The translational energy distributions for each one of these steps have been derived using the forward convolution technique. These energy distributions reveal the exit barriers and other constraints on the potential energy surfaces that lead to the above stated products.
In analogy to pressure-driven gradient techniques in high-performance liquid chromatography, a system has been developed for delivering electroosmotically driven solvent gradients for capillary electrochromatography (CEC). Dynamic gradients with submicroliter per minute flow rates are generated by merging two electroosmotic flows that are regulated by computer-controlled voltages. These flows are delivered by two fused-silica capillary arms attached to a T-connector, where they mix and then flow into a capillary column that has been electrokinetically packed with 3-μm reversed-phase particles. The inlet of one capillary arm is placed in a solution reservoir containing one mobile phase, and the inlet of the other is placed in a second reservoir containing a second mobile phase. Two independent computer-controlled, programmable, high-voltage power supplies (0−50 kV)one providing an increasing ramp and the other providing a decreasing rampare used to apply variable high-voltage potentials to the mobile phase reservoirs to regulate the electroosmotic flow in each arm. The ratio of the electroosmotic flow rates between the two arms is changed with time according to the computer-controlled voltages to deliver the required gradient profile to the separation column. Experiments were performed to confirm the composition of the mobile phase during a gradient run and to determine the change of the composition in response to the programmed voltage profile. To demonstrate the performance of electroosmotically driven gradient elution in CEC, a mixture of 16 polycyclic aromatic hydrocarbons was separated in less than 90 min. This gradient technique is expected to be well-suited for generating not only solvent gradients in CEC but also other types of gradients, such as pH and ionic strength gradients, in capillary electrokinetic separations and analyses.
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