Biomedical subjects

Reggie S Spaulding

Publications and source records attributed to Reggie S Spaulding.

4 recordsLinked to original sources

PubMed2005

Analysis and sequencing of the active-site peptide from native and organophosphate-inactivated acetylcholinesterase by electrospray ionization, quadrupole/time-of-flight (QTOF) mass spectrometry.

A method to identify and sequence recombinant mouse acetylcholinesterase (rMoAChE) including the native and organophosphate-modified active-site peptides was developed using capillary liquid chromatography with electrospray ionization, quadrupole/time-of-flight mass spectrometry. Addition of 2-propanol to the reversed-phase gradient system and a decreased gradient slope improved the peptide resolution and the signal of the active-site peptide. The highest protein coverage and active-site peptide signal were achieved when the rMoAChE:chymotrypsin ratio of 5:1 was used with digestion at 37 degrees C. rMoAChE and the active-site peptide were identified and sequenced from chymotryptic digests of native, methyl paraoxon-, and ethyl paraoxon-inactivated rMoAChE showing unequivocally that the exact modification site was the active-site serine.

Acetylcholinesterase↗

PubMed2004

The synaptic vesicle proteome: a comparative study in membrane protein identification.

A proteomic analysis of the synaptic vesicle was undertaken to obtain a better understanding of vesicle regulation. Synaptic vesicles primarily consist of integral membrane proteins that are not well resolved on traditional isoelectric focusing/two-dimensional gel electrophoresis (IEF/2-DE) gels and are resistant to in-gel digestion with trypsin thereby reducing the number of peptides available for mass spectrometric analysis. To address these limitations, two complementary 2-DE methods were investigated in the proteome analysis: (a) IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) for resolution of soluble proteins and, (b) Benzyl hexadecyl ammonium chloride/SDS-PAGE (16-BAC/SDS-PAGE) for resolution of integral membrane proteins. The IEF/SDS-PAGE method provided superior resolution of soluble proteins, but could only resolve membrane proteins with a single transmembrane domain. The 16-BAC/SDS-PAGE method improved separation, resolution and identification of integral membrane proteins with up to 12 transmembrane domains. Trypsin digestion of the integral membrane proteins was poor and fewer peptides were identified from these proteins. Analysis of both the peptide mass fingerprint and the tandem mass spectra using electrospray ionization quadrupole-time of flight mass spectrometry led to the positive identification of integral membrane proteins. Using both 2-DE separation methods, a total of 36 proteins were identified including seven integral membrane proteins, 17 vesicle regulatory proteins and four proteins whose function in vesicles is not yet known.

Cell Membrane↗

PubMed2002

Ambient air measurement of acrolein and other carbonyls at the Oakland-San Francisco Bay Bridge toll plaza.

Interest in ambient concentrations of acrolein and other alpha,beta-unsaturated aldehydes and dicarbonyls (e.g., crotonaldehyde, methyl glyoxal, glyoxal, malonaldehyde (malondialdehyde)) is growing because either they exist at high levels in motor vehicle emissions or they arise from photooxidation of other hydrocarbons emitted from mobile sources. In addition, their mutagenic, genotoxic, or carcinogenic properties are well-established, and the results of a dispersion-modeling study regarding the health risks posed by the 188 hazardous air pollutants in California attributes the highest noncancer risk to exposure to acrolein. Such modeling studies, conducted by the U.S. Environmental Protection Agency (U.S. EPA), also predict median ambient air concentrations of acrolein higher than 0.06 microg/m3, the chronic inhalation reference exposure level stipulated by the California Office of Environmental Health Hazard Assessment in counties surrounding the Oakland-San Francisco Bay Bridge. We measured acrolein and other potentially toxic carbonyls in air sampled at the San Francisco Bay Bridge toll plaza during rush hour traffic, which may be considered a "worst case scenario" for outdoor airborne carbonyls. We identified 36 carbonyls in the sample extracts, including 14 saturated aliphatic carbonyls, six unsaturated carbonyls, four aromatic carbonyls, six dicarbonyls, and six hydroxy carbonyls. Structural information to support tentative identification of carbonyls and hydroxycarbonyls was obtained by using a method that involves O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) and PFBHA/bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization in concert with gas chromatography/ion trap mass spectrometry. Most notably, we report for the first time the presence of malonaldehyde in the ambient atmospheric environment. A relatively linear relationship between retention time and the molecular weight of the derivatives was established to assist in obtaining structural information about chemicals for which authentic standards are not readily available. Levels of acrolein exceeded the California reference exposure level during morning rush hour traffic. The measured values, however, were significantly lower than estimates of county-wide average acrolein concentrations predicted by a U.S. EPA modeling study based on 1996 data. Successful regulatory efforts such as the introduction of reformulated gasoline, together with the advancement of new catalysts and fleet turnover throughout the 1990s, are likely to account for part of the gap between our determination and the 1996 levels.

Acrolein↗

PubMed2002

Optimization of a mist chamber (Cofer scrubber) for sampling water-soluble organics in air.

While the atmospheric fate and transport of biogenic and anthropogenic hydrocarbons has been extensively studied, little is known about the behavior of first-, second-, and third generation photo-oxidation products that arise from OH radical oxidation of the parent species. The results of chamber experiments establish that *OH oxidation of biogenic and anthropogenic hydrocarbons yields carbonyls, dicarbonyls, hydroxycarbonyls, and keto-acids. However, little is known about the generation and fate of these products in the ambient atmospheric environment. This is changing because of the advent of methods that rely on 0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) derivatization of carbonyls in concert with gas chromatography/ion trap mass spectrometry. Such methods provide the means to identify and quantify water-soluble organics, which historically have been difficult to measure. A limitation of existing sampling methods, however, is the use of devices that require low flow rates (0.5-1 L min(-1)). Accordingly, long sampling times (3-4 h) are needed to obtain pptv-ppbv detection limits. The mist chamber is an attractive device because of the high flow rates (25-70 L min(-1)) compatible with its use. Herein, we evaluate a mist chamber using a flow rate of 25-30 L min(-1) to provide short (10 min) sampling times and pptv limits of detection. The results establish a relationship between the Henry's law constant (KH) and the collection efficiency and demonstrate the suitability of the method to measure analytes with KH > or = 10(3) M atm(-1). Adjusting the pH, adding quaternary ammonium salts, or decreasing the temperature of the collecting solution in the mist chamber did not significantly affect the collection efficiency. We tested the method by sampling photooxidation products of isoprene (glyoxal, methylglyoxal, hydroxyacetone, and glycolaldehyde) in the Blodgett Forest, CA. This is the first report of a study the employs the mist chamberto sample hydroxycarbonyls. The accuracy and the reproducibility of the method were evaluated by the analysis of duplicate samples and field spikes. The mean recovery of field spikes was > or =80%, and the relative standard deviation was < or =22% between duplicate measurements. The detection limits were 48, 15, 7.7, and 2.7 pptv for glycolaldehyde, hydroxyacetone, methylglyoxal, and glyoxal, respectively. This work demonstrates the power of the mist chamber in concert with PFBHA derivatization and mass spectrometry to measure pptv concentrations of water-soluble organics with a sampling time of 10 min.

Air Movements↗