PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-5 (5)
 

Clipboard (0)
None

Select a Filter Below

Journals
Authors
more »
Year of Publication
Document Types
1.  QuEChERS-based approach toward the analysis of two insecticides, methomyl and aldicarb, in blood and brain tissue 
QuEChERS has been widely utilized for the analysis of pesticides in produce, but it has not been as widely used in clinical test specimens, especially for smaller, sub-gram sample sizes. This study describes the application of a miniaturized QuEChERS methodology toward the analysis of two insecticides, methomyl and aldicarb, in guinea pig blood and brain tissue. Matrix effects and absolute recoveries were investigated for both analytes in the two matrices. While the matrix effects of methomyl in both matrices were minimal at most levels (i.e., from −20% to 20%), aldicarb experienced signal suppression under the described conditions (mean of −47%). However, the matrix effects were not cause for concern due to the sensitivity of the method and the use of matrix-matched standards. The precision and accuracy of the method were excellent over a range of concentrations that spanned three orders of magnitude. The limits of detection (LOD) for both carbamates were determined to be 0.1 ng mL−1 in blood and 0.2 ng g−1 in brain. Other validation parameters, such as linearity, accuracy, precision, and recovery, were also satisfactory in the blood and brain tissue. This method was demonstrated to be sensitive and reproducible, and it should be applicable to the analysis of a wide range of compounds of interest in sub-gram- and sub-milliliter-sized clinical and toxicology specimens.
doi:10.1039/C4AY02137A
PMCID: PMC4287231  PMID: 25580162
2.  Thermodynamic Analysis of Protein-Ligand Binding Interactions in Complex Biological Mixtures using the Stability of Proteins from Rates of Oxidation (SPROX) Method 
Nature protocols  2012;8(1):148-161.
The detection and quantitation of protein-ligand binding interactions is critical in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g. drugs like tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (iTRAQ) with a covalent labeling technique, termed Stability of Proteins from Rates of Oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide-mediated oxidation of methionine residues as a function of the chemical denaturant (e.g. guanidine Hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to detection of both the on- and off-target effects of ligand binding.
doi:10.1038/nprot.2012.146
PMCID: PMC3717606  PMID: 23257983
Protein-ligand; protein-drug; interaction; mass spectrometry; iTRAQ; shotgun proteomics; hydrogen peroxide; H2O2; methionine oxidation; denaturant; guanidine; urea; protein folding/unfolding
3.  Thermodynamic Analysis of Protein-Ligand Interactions in Complex Biological Mixtures using a Shotgun Proteomics Approach 
Journal of proteome research  2011;10(11):4948-4958.
Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme co-factor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.
doi:10.1021/pr200403c
PMCID: PMC3208786  PMID: 21905665
Protein-ligand binding; resveratrol; NAD+; methionine oxidation; iTRAQ; SPROX
4.  Stable Isotope Labeling Strategy for Protein–Ligand Binding Analysis in Multi-Component Protein Mixtures 
Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein–ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein–ligand binding interactions. The protocol utilizes an H216O2 and H218O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein–ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique’s ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein–protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).
doi:10.1007/s13361-010-0060-1
PMCID: PMC3085011  PMID: 21472561
Protein folding; Ligand binding; Thermodynamics; Covalent labeling; Quantitative proteomics; Cyclophilin A; Cyclosporin A; Calcineurin
5.  Discovery of Novel Cyclophilin A Ligands Using an H/D Exchange- and Mass Spectrometry-Based Strategy 
Journal of biomolecular screening  2010;15(9):1051-1062.
Cyclophilin A (CypA) is an overexpressed protein in lung cancer tumors and as a result is a potential therapeutic and diagnostic target. Here we utilize an H/D exchange- and MALDI mass spectrometry-based assay, termed single-point SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), to screen two chemical libraries, including the 1280-compound LOPAC library and the 9600 compound DIVERSet library, for binding to CypA. This work represents the first application of single-point SUPREX using a pooled ligand approach, which we demonstrate is capable of screening rates as fast as six seconds/ligand. The false positive and false negative rates determined in the current work using a set of control samples were 0% and 9%, respectively. A false positive rate of 20% was found in screening the actual libraries. Eight novel ligands to CypA were discovered including: 2-(α-naphthoyl)ethyltrimethyl-ammonium iodide, (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, 3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one, cis-diammineplatinum (II) chloride, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, N-(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione, and 4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[5.2.1.0~2,6~]dec-8-ene-3,5-dione. These compounds, which had moderate binding affinities to CypA (i.e., Kd values in the low micromolar range), provide new molecular scaffolds that might be useful in the development of CypA targeted diagnostic imaging or therapeutic agents for lung cancer.
doi:10.1177/1087057110382775
PMCID: PMC3197229  PMID: 20855564
Cyclophilin A; Matrix-Assisted Laser Desorption/Ionization; amide H/D exchange; high-throughput screening

Results 1-5 (5)