Development and validation of a quantitative assay for the measurement of two HIV-fusion inhibitors, enfuvirtide and tifuvirtide, and one metabolite of enfuvirtide (M-20) in human plasma by liquid chromatography-tandem mass spectrometry

A method for the quantification of two peptide HIV-1 fusion inhibitors (enfuvirtide, T-20 and tifuvirtide, T-1249) and one metabolite of enfuvirtide (M-20) in human plasma has been developed and validated, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). The analytes were extracted from plasma by solid-phase extraction (SPE) on vinyl-copolymer cartridges. Chromatographic separation of the peptides was performed on a Symmetry 300 C(18) column (50mmx2.1mm I.D., particle size 3.5 microm), using a water-acetonitrile gradient containing 0.25% (v/v) formic acid. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for peak detection. Deuterated (d60) enfuvirtide and (d50) tifuvirtide were used as internal standards. The assay was linear over a concentration range of 20-10,000 ng/ml for enfuvirtide and tifuvirtide and of 20-2000 ng/ml for M-20. Intra- and inter-assay precisions and deviations from the nominal concentrations were 相似文献   

Simultaneous determination of lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma using high-performance liquid chromatography coupled with electrochemical detection

A fast, simple, and a reliable high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method for the assessment of lipoic acid (LA) and dihydrolipoic acid (DHLA) in plasma was developed using naproxen sodium as an internal standard (IS) and validated according to standard guidelines. Extraction of both analytes and IS from plasma (250 μl) was carried out with a single step liquid-liquid extraction applying dichloromethane. The separated organic layer was dried under stream of nitrogen at 40°C and the residue was reconstituted with the mobile phase. Complete separation of both compounds and IS at 30°C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 9 min using acetonitrile: 0.05 M phosphate buffer (pH 2.4 adjusted with phosphoric acid) (52:48, v/v) as a mobile phase pumped at flow rate of 1.5 ml min(-1) using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification for lipoic acid were 500 pg/ml and 3 ng/ml, and for dihydrolipoic acid were 3 ng/ml and 10 ng/ml, respectively. The absolute recoveries of lipoic acid and dihydrolipoic acid determined on three nominal concentrations were in the range of 93.40-97.06, and 93.00-97.10, respectively. Similarly coefficient of variations (% CV) for both intra-day and inter-day were between 0.829 and 3.097% for lipoic acid and between 1.620 and 5.681% for dihydrolipoic acid, respectively. This validated method was applied for the analysis of lipoic acid/dihydrolipoic acid in the plasma of human volunteers and will be used for the quantification of these compounds in patients with oxidative stress induced pathologies.     

Optimization of digestion parameters for protein quantification

We present a rapid and efficient in-solution enzymatic digestion protocol suitable for mass spectrometry-based absolute protein quantification techniques. The digestion method employs RapiGest SF (an acid-labile surfactant), an excess amount of modified trypsin (enzyme-to-substrate ratio of 2.5:1), and an incubation time of 2 h. No reduction/alkylation reagents are used. Digestion parameters were varied systematically to monitor their effect on rate and completeness of digestion. To demonstrate the general applicability of the method, the optimization was done using a viral hemagglutinin (HA) as a model protein and then applied to ricin, a potent protein toxin extracted from the castor bean (Ricinus communis). The parameters that were optimized included incubation time, concentration of RapiGest SF, enzyme-to-substrate ratio, and incubation temperature. The optimization was done by comparing the yields from two protein-specific peptides originating from two different sites of the HA protein. The analysis was performed by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode using isotopically labeled peptide standards for quantification.     

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Papain fragmentation of the (Na+,K+)-ATPase beta subunit reveals multiple membrane-bound domains

Purified dog kidney (Na+,K+)-ATPase was reacted with tritiated sodium borohydride after treatment with neuraminidase and galactose oxidase. This procedure did not affect the ATPase activity of the enzyme, and all of the covalently bound radioactivity was found in the beta subunit (Mr 54 000). Papain digestion of the tritiated enzyme produced two labeled fragments of Mr 40 000 and 16 000. Further proteolysis generated an Mr 31 000 peptide from the larger fragment. Unlike the tryptic and chymotryptic sites of the alpha subunit, the sites of papain hydrolysis were insensitive to conformations of the (Na+,K+)-ATPase. Determination of the NH2-terminal sequences was used to arrange the fragments within the linear map of the beta chain. Finally, none of the labeled peptides was released from the membrane under nondenaturing conditions. These results are consistent with a model of the beta subunit containing a 40 000-dalton NH2-terminal piece and a 16 000-dalton COOH-terminal piece. Both fragments have extracellularly exposed carbohydrate and at least one membrane-bound domain.     

The calmodulin-binding domain on microtubule-associated protein 2

Microtubule-associated protein 2 (MAP2) binds calmodulin with a stoichiometry approaching 1-1.5 mol of calmodulin/mol of MAP2 in the presence of calcium ion. The calmodulin-binding domain(s) of MAP2 were probed by cross-linking 125I-calmodulin with partially digested MAP2, by limited digestion of the preformed 125I-calmodulin-MAP2 adduct, and by cross-linking 125I-calmodulin with the projection- and assembly-promoting portions of MAP2. Cross-linking 125I-calmodulin with partially digested MAP2 resulted in radioactive adducts of approximately 300, approximately 235, approximately 205, approximately 58, and approximately 40 kDa. The radioactive adducts with smaller molecular mass became prominent with increasing time of digestion concomitant with loss of those with higher molecular size. Limited chymotryptic digestion of preformed 125I-calmodulin-MAP2 adducts also produced a approximately 58-kDa radioactive band followed later by a approximately 40-kDa band. Brief chymotryptic digestion and subsequent centrifugation of microtubules preformed with pure tubulin and MAP2 permitted separation of microtubule-bound MAP2 fragments (molecular mass = approximately 215, approximately 180, and approximately 36 kDa) from unbound fragments (molecular mass = approximately 240, approximately 180, and approximately 140 kDa). 125I-Calmodulin cross-linked only with the microtubule-bound MAP2 fragments (forming mainly the approximately 58-kDa adduct) and not with unbound MAP2 fragments. Since the apparent molecular size of calmodulin is approximately 21 kDa on these sodium dodecyl sulfate-polyacrylamide gels, the results indicate that partial digestion of MAP2 by chymotrypsin produces a approximately 37-kDa fragment which can be further degraded to a approximately 20-kDa fragment. The approximately 37-kDa fragment that is labeled corresponds to the previously identified assembly-promoting fragment that attaches to the microtubule.     

Structural and antigenic characterization of a species- and promastigote-specific Leishmania mexicana amazonensis membrane protein

A Leishmania mexicana amazonensis promastigote membrane glycoprotein (Mr 46,000) expressing the species-specific and promastigote-specific epitope of monoclonal antibody IX 2H7-E10(M-2) has been purified to homogeneity, and studies have been made to determine the minimum peptide fragment that retained antigenic activity. Peptide mapping experiments performed with the metabolically labeled or surface radioiodinated protein illustrated its highly folded nature and marked resistance to proteolytic digestion. The M-2 epitope was readily destroyed by limited proteolysis and/or reduction and alkylation, indicating disulfide bond involvement in its formation by at least the secondary protein structure. The stability of approximately half of the molecular mass of the protein (46kDa/M-2) was also dependent on disulfide bonding. Enzymic digests under various conditions generated a glycopolypeptide (Mr 22,000 to 27,000), extremely resistant to further enzymic digestion, that was the dominant immunogenic portion of the purified protein recognized by a specific rabbit heteroserum. No smaller or larger fragments were antigenic. Data obtained by using the radioiodinated hydrophobic probe 3-(trifluoromethyl)-3-([m-125]iodophenyl)-diazirine ([125I]TID) indicate that 46kDa/M-2 is an integral membrane protein with a component polypeptide (Mr 23,000 to 27,000), highly resistant to further enzymic cleavage and containing sequences within the external promastigote membrane. Data indicate that the [125I]TID-labeled fragment is identical to the immunodominant fragment. We suggest that hydrophobic interactions maintain the integrity of this fragment as amino acids within it fold through the parasite membrane.     

Dihydrofolate reductase: the amino acid sequence of the enzyme from a methotrexate-resistant mutant of Escherichia coli.

The determination of the amino acid sequence of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) from a mutant of Escherichia coli B is described. The 159 residues were positioned by automatic Edman degradation of the whole protein, of the reduced and alkylated cyanogen bromide fragments, and of selected tryptic, chymotryptic, and thermolytic digestion products. An N-bromosuccinimide produced fragment of the largest cyanogen bromide peptide was also used in the sequence determination.     

Detection of human butyrylcholinesterase-nerve gas adducts by liquid chromatography-mass spectrometric analysis after in gel chymotryptic digestion

To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis.     

Cleavage of pyridoxal kinase into two structural domains: Kinetics of proteolysis monitored by emission anisotropy

Two sulfhydryl residues/dimer of pyridoxal kinase react with iodoacetamide fluoresceine (IAF) to yield catalytically active species. Limited chymotryptic digestion of IAF pyridoxal kinase resulted in the release of two fragments of 24 and 16 KDA. One of the fragments (16 KDA) is labeled with IAF. After complete tryptic digestion of IAF-pyridoxal kinase, only one peptide labeled with IAF was separated by reverse-phase HPLC and its amino acid sequence determined by automated Edman degradation. The kinetics of chymotryptic cleavage of IAF-pyridoxal kinase was monitored by steady-state emission anisotropy measurements. Analysis of the kinetic results revealed that the rate of proteolysis is significantly reduced by the substrate pyridoxal (0.2 mM). ATP (1 mM) does not influence the rate of proteolysis. The technique of emission anisotropy was also applied to monitor the effect of viscosity on the rate of proteolysis. A kinetic model is proposed to explain the mechanism of limited proteolysis. The model is based on the assumption that unfolding of the native conformation of the protein-substrate complex plays a dominant role in proteolysis.     

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.     

Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase

Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.     

Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry

Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.     

Molecular modeling and experimental approaches toward designing a minimalist protein having Fc-binding activity of Staphylococcal protein A

Protein A (PA), a cell wall constituent of Staphylococcus aureus, has got the unique property of binding with the Fc fragment of IgG from various species. The sequence data indicate five highly homologous Fc-binding regions in protein A. Computer sequence analysis provided the tryptic and chymotryptic fragments of IgG-binding domains of protein A. Molecular modeling in conjunction with molecular mechanical calculation has been used to search for the smallest possible proteolytic fragments of PA, still retaining Fc-binding activity. A 20-residue peptide (typtic fragment) and a 16-residue peptide (chymotryptic fragment) have been indicated, by molecular modeling studies, to possess IgG-binding affinity comparable to that of the B domain of Protein A. Binding of a 20-residue peptide has been substantiated experimentally by immunoprecipitation, capillary electrophoresis, and circular dichroism spectroscopic analyses.     

IQcat: multiplexed protein quantification by isoelectric QconCAT

Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by pI to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (～2 weeks), inexpensive and scalable purification of arg/lys labeled IQcat standards in the Escherichia coli auxotroph AT713. With this pipeline, we assess the fidelity of IQcat-based absolute quantification for ten yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.     

Amino acid sequence of rabbit skeletal muscle myosin. 50-kDa fragment of the heavy chain

The amino acid sequence of the 50-kDa fragment that is released by limited tryptic digestion of the head portion of rabbit skeletal muscle myosin was determined by analysis and alignment of sets of peptides generated by digestion of the fragment at arginine or methionine residues. This fragment contains residues 205-636 of the myosin heavy chain; among the residues of particular interest in this fragment are N epsilon-trimethyllysine, one of four methyl-amino acids in myosin, and Ser-324, which is photoaffinity labeled by an ATP analogue (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995). Combination of this sequence with those of the 23- and 20-kDa fragments yields an 809-residue sequence that constitutes most of the heavy chain of chymotryptic S-1 of this myosin.     

Binding site for vitamin K-dependent protein S on complement C4b-binding protein

Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.     

Quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry

The method describes quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS/MS). Samples were screened by enzyme-linked immunosorbent assay (ELISA) and only those samples presumptively declared positive were subjected to quantification and confirmation for the presence of flunixin by this method. The method is also readily adaptable to instrumental screening for the analyte. Flunixin was recovered from plasma by liquid-liquid extraction (LLE). The sample was diluted with 2 ml saturated phosphate buffer (pH 3.10) prior to LLE. The dried extract was reconstituted in acetonitrile:water:formic acid (50:50:0.1, v/v/v) and subsequently analyzed on a Q-TOF tandem mass spectrometer (Micromass) operated under electrospray ionization positive ion mode. The concentration of flunixin was determined by the internal standard (IS) calibration method using the peak area ratio with clonixin as the IS. The limits of detection (LOD) and quantification (LOQ) for flunixin in equine plasma were 0.1 and 1 ng/ml, respectively, whereas the limit of confirmation (LOC) was 2.5 ng/ml. The qualifying ions for the identification of flunixin were m/z 297 [M+H](+), 279 (BP), 264, 259, 239 and those for clonixin (IS) were m/z 263 [M+H](+), 245 (BP) and 210. The measurement uncertainty about the result was 8.7%. The method is simple, sensitive, robust and reliably fast in the quantification and confirmation of flunixin in equine plasma. Application of this method will assist racing authorities in the enforcement of tolerance plasma concentration of flunixin in the racehorse on race day.     

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [3H]dexamethasone 21-mesylate ([3H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing, non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [3H]DM-labeled tryptic fragments resolved two (pI congruent to 5.7 and 7.0) 26.5-kDa and two (pI congruent equal to 5.7 and 6.8) 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [3H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [3H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.     

Validation of a simple liquid chromatography-tandem mass spectrometric method for the determination of propiverine hydrochloride and its N-oxide metabolite in human plasma

A simple high-performance liquid chromatography (HPLC)-tandem mass spectrometric method has been developed for determination of propiverine hydrochloride and its metabolite, propiverine N-oxide (M-1) in human plasma using stable isotopes, propiverine hydrochloride-d10 and M-1-d10, as internal standards. The analytes were extracted with dichloromethane from 0.2 ml of plasma in neutral condition (pH 7.0) and separated by HPLC on a C18 reversed-phase column using methanol-1% acetic acid (50:50) as a mobile phase, and detected using positive electrospray ionization in selected reaction monitoring (SRM) mode. The method was validated over a concentration range of 2-500 ng/ml for propiverine hydrochloride and 4-1000 ng/ml for M-1 using 0.2 ml of human plasma per assay. The method developed was successfully applied to analysis of propiverine hydrochloride and M-1 in clinical studies.