Urinary Creatinine Concentrations in the U.S. Population: Implications for Urinary Biologic Monitoring Measurements-0

<p><b>Copyright information:</b></p><p>Taken from "Urinary Creatinine Concentrations in the U.S. Population: Implications for Urinary Biologic Monitoring Measurements"</p><p>Environmental Health Perspectives 2004;113(2):192-200.</p><p>Published online 23 Sep 2004</p><p>PMCID:PMC1277864.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Optimization of the linear quantification range of an online trypsin digestion coupled liquid chromatography–tandem mass spectrometry (LC–MS/MS) platform

<p>Tandem mass spectrometry (MS/MS)-based proteomic workflows with a bottom-up approach require enzymatic digestion of proteins to peptide analytes, usually by trypsin. Online coupling of trypsin digestion of proteins, using an immobilized enzyme reactor (IMER), with liquid chromatography (LC) and MS/MS is becoming a frequently used approach. However, finding IMER digestion conditions that allow quantitative analysis of multiple proteins with wide range of endogenous concentration requires optimization of multiple interactive parameters: digestion buffer flow rate, injection volume, sample dilution, and surfactant type/concentration. In this report, we present a design of experiment approach for the optimization of an integrated IMER-LC–MS/MS platform. With bovine serum albumin as a model protein, the digestion efficacy and digestion rate were monitored based on LC–MS/MS peak area count versus protein concentration regression. The optimal parameters were determined through multivariate surface response modeling and consideration of diffusion controlled immobilized enzyme kinetics. The results may provide guidance to other users for the development of quantitative IMER-LC–MS/MS methods for other proteins.</p

Quantification of Botulinum Neurotoxin Serotypes A and B from Serum Using Mass Spectrometry

Botulinum neurotoxins (BoNT) are the deadliest agents known. Previously, we reported an endopeptidase activity based method (Endopep-MS) that detects and differentiates BoNT serotypes A–G. This method uses serotype specific monoclonal antibodies and the specific enzymatic activity of BoNT against peptide substrates which mimic the toxin’s natural target. Cleavage products from the reaction are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We have now developed a multiple reaction monitoring method to quantify the biological activity of BoNT serotypes A (BoNT/A) and B (BoNT/B) present in 0.5 mL of serum using electrospray mass spectrometry. The limit of quantification for each serotype is 1 mouse intraperitoneal lethal dose (MIPLD₅₀) corresponding to 31 pg of BoNT/A and 15 pg of BoNT/B in this study. This method was applied to serum from rhesus macaques with inhalational botulism following exposure to BoNT/B, showing a maximum activity of 6.0 MIPLD₅₀/mL in surviving animals and 653.6 MIPLD₅₀/mL in animals that died in the study. The method detects BoNT/B in serum 2–5 h after exposure and up to 14 days. This is the first report of a quantitative method with sufficient sensitivity, selectivity, and low sample size requirements to measure circulating BoNT activity at multiple times during the course of botulism

Quantification of Metabolites for Assessing Human Exposure to Soapberry Toxins Hypoglycin A and Methylenecyclopropylglycine

Ingestion of soapberry fruit toxins hypoglycin A and methylenecyclopropylglycine has been linked to public health challenges worldwide. In 1976, over 100 years after Jamaican vomiting sickness (JVS) was first reported, the cause of JVS was linked to the ingestion of the toxin hypoglycin A produced by ackee fruit. A structural analogue of hypoglycin A, methylenecyclopropylglycine (MCPG), was implicated as the cause of an acute encephalitis syndrome (AES). Much of the evidence linking hypoglycin A and MCPG to these diseases has been largely circumstantial due to the lack of an analytical method for specific metabolites. This study presents an analytical approach to identify and quantify specific urine metabolites for exposure to hypoglycin A and MCPG. The metabolites are excreted in urine as glycine adducts methylenecyclopropylacetyl-glycine (MCPA-Gly) and methylenecyclopropylformyl-glycine (MCPF-Gly). These metabolites were processed by isotope dilution, separated by reverse-phase liquid chromatography, and monitored by electrospray ionization tandem mass spectrometry. The analytical response ratio was linearly proportional to the concentration of MCPF-Gly and MCPA-Gly in urine from 0.10 to 20 μg/mL with a correlation coefficient of <i>r</i> > 0.99. The assay demonstrated accuracy ≥80% and precision ≤20% RSD across the calibration range. This method has been applied to assess exposure to hypoglycin A and MCPG as part of a larger public health initiative and was used to provide the first reported identification of MCPF-Gly and MCPA-Gly in human urine

Sequence alignment of known BoNT subtypes in the range of Y750 to E757 of BoNT/A.

<p>Residues that play an important role in 4E17.1 binding are bolded and underlined. Dissociation rates (K_D) in pM of BoNT with mAb 4E17.1 are also listed. NB indicates no binding was observed and ND indicates that the K_D was not determined. Each BoNT is also identified by strain tested where appropriate. Equilibrium dissociation constant (K_D) were measured by flow fluorimetry in a KinExA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012237#pone.0012237-GarciaRodriguez1" target="_blank">[17]</a>.</p

Mass spectra of the Endopep-MS BoNT/A reactions with /A1 (1A), /A2 (1B), /A3 (1C), /A4 (1D), or no toxin (1E) extracted using the 4E17.1 antibody-coated beads.

<p>The peptide cleavage products indicating BoNT/A are <i>m/z</i> 1197.8 and 1699.9; the peptide substrate is present at <i>m/z</i> 2878.7.</p

Mass spectra of the Endopep-MS BoNT/F reactions with /F1 (4A), /F6 (4B), /F2 (4C), or no toxin (4D) extracted using the 4E17.1 antibody-coated beads.

<p>The peptide cleavage products indicating BoNT/F are <i>m/z</i> 1345.2 and 3167.8 and the peptide substrate is present at <i>m/z</i> 4497.5.</p

Amino acid sequence of peptides used in and produced by the Endopep-MS method.

<p>The observed <i>m/z</i> of each peptide is also included.</p

Mass spectra of the Endopep-MS BoNT/E reactions with /E1 (3A), /E2 (3B), /E3 (3C), /E4 (3D), or no toxin (3E) extracted using the 4E17.1 antibody-coated beads.

<p>The peptide cleavage products indicating BoNT/E are <i>m/z</i> 1136.6 and 2923.6 and the peptide substrate is present at <i>m/z</i> 4041.1.</p

Mass spectrum of the Endopep-MS reaction of the simultaneous extraction of BoNT/A, /B, /E, and /F with monoclonal antibody 4E17.1 (6A) or the single extraction of BoNT/A (6B), /B(6C), /E (6D), or /F (6E) with serotype-specific antibodies.

<p>Cleavage products indicating those four BoNTs are marked.</p