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S P Gygi

Publications and source records attributed to S P Gygi.

34 records · Page 2Linked to original sources
PubMed1999

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.

Affinity Labels↗
PubMed1999

Correlation between protein and mRNA abundance in yeast.

We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast Saccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243-251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.

Codon↗
PubMed1998

An integrated microfluidics-tandem mass spectrometry system for automated protein analysis.

We describe an integrated analytical system consisting of a microfluidics device micromachined using photolithography/etching technology, a panel of computer-controlled high-voltage relays, and an electrospray ionization tandem mass spectrometer. Movement of solvents and samples on the device and off the device to the mass spectrometer was achieved by directed electroosmotic pumping induced by the activation of a suitable constellation of high-voltage relays. The system was used for the sequential automated analysis of protein digests. We demonstrate low femtomole per microliter sensitivity of detection and compatibility of the system with the automated analysis of proteins separated by two-dimensional gel electrophoresis.

Amino Acid Sequence↗
PubMed1998

Electrophoresis combined with novel mass spectrometry techniques: powerful tools for the analysis of proteins and proteomes.

Analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). A series of different techniques, consisting of automated high performance liquid chromatography (HPLC)-MS/MS, capillary-HPLC-MS/MS, and solid phase extraction (SPE)-capillary zone electrophoresis (CZE)-MS/MS, are described in the context of the identification of high pmol to the low fmol amounts of proteins. Application of these powerful new tools for the analysis of proteins on a large proteome-wide scale is presented. Furthermore, the combination of orthogonal separation techniques, such as immobilized metal affinity chromatography (IMAC) with SPE-CZE-MS/MS, and IMAC followed by HPLC, and by SPE-CZE-MS/MS, are presented for the detailed investigation of post-translational modifications of specific proteins.

Amino Acid Sequence↗
PubMed1998

Proteome analysis: biological assay or data archive?

In this review we examine the current state of proteome analysis. There are three main issues discussed: why it is necessary to study proteomes; how proteomes can be analyzed with current technology; and how proteome analysis can be used to enhance biological research. We conclude that proteome analysis is an essential tool in the understanding of regulated biological systems. Current technology, while still mostly limited to the more abundant proteins, enables the use of proteome analysis both to establish databases of proteins present, and to perform biological assays involving measurement of multiple variables. We believe that the utility of proteome analysis in future biological research will continue to be enhanced by further improvements in analytical technology.

Animals↗
PubMed1998

Incorporation of drugs for the treatment of substance abuse into pigmented and nonpigmented hair.

Hair analysis for drugs may be useful for the long-term monitoring of recidivism and treatment compliance. L-alpha-Acetylmethadol, buprenorphine, and methadone are drugs that are used for the treatment of substance abuse. The purpose of this study was to study the relationship between dose, plasma concentration, hair concentration, and hair pigmentation for these compounds and their major metabolites in an animal model. Male Long-Evans rats received either L-alpha-acetylmethadol (1 and 3 mg/kg; n = 6), buprenorphine (1 and 3 mg/kg; n = 5), or methadone (4 and 8 mg/kg; n = 5) by intraperitoneal injection daily for 5 days. Fourteen days after beginning drug administration, newly grown hair was collected and analyzed for either L-alpha-acetylmethadol and two metabolites (L-alpha-acetyl-N-normethadol and L-alpha-acetyl-N,N-dinormethadol), methadone and two metabolites (D,L-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium and D,L-2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline), or buprenorphine and one metabolite (norbuprenorphine). The plasma time course (AUC) for each compound was also determined after a single administration of each drug at the specified doses. There was an approximate dose-dependent increase in measured hair concentration of each parent drug in pigmented hair. The concentrations of L-alpha-acetylmethadol, methadone, and buprenorphine in nonpigmented hair were significantly less than that measured in pigmented hair at either the high or low dose. The metabolites L-alpha-acetyl-N-normethadol and D,L-2-ethyl-1,5dimethyl-3,3-diphenylpyrrolinium were detected at lower concentrations than their respective parent compounds (L-alpha-acetylmethadol or methadone) in pigmented hair. However, the L-alpha-acetyl-N,N-dinormethadol metabolite concentrations in pigmented hair were significantly greater than those of the parent drug after either the low or the high L-alpha-acetylmethadol dose. These data demonstrate that L-alpha-acetylmethadol, methadone, buprenorphine, and metabolites are distributed into hair in a dose-related manner with a preference for pigmented hair.

Animals↗
PubMed1997

A comparison of phenobarbital and codeine incorporation into pigmented and nonpigmented rat hair.

Drugs and endogenous compounds circulating in the blood may ultimately become incorporated into a growing hair shaft. Hair analysis for drugs of abuse is a growing field in the area of forensic and clinical toxicology. However, the underlying principles that govern drug incorporation into hair are not known. In this study, we examined the incorporation of a weak acid, phenobarbital, and a weak base, codeine, into Sprague-Dawley (SD) rat hair. Codeine or phenobarbital was administered to male SD rats at 40 mg/kg/day for 5 days by intraperitoneal (ip) injection. Hair was collected from the back 14 days after beginning the 5-day dosing protocol and analyzed by gas chromatography/mass spectrometry (GC/MS) for codeine and phenobarbital. The time-courses of phenobarbital and codeine in plasma were also obtained after a single ip injection (40 mg/kg). Concentrations of codeine and phenobarbital in SD hair samples were 0.98 +/- 0.10 and 17.01 +/- 1.40 ng/mg hair. respectively. The areas under the curve (AUC) of plasma concentration versus time for codeine and phenobarbital were 1.58 and 414.50 micrograms h/microL, respectively. Notwithstanding the greater phenobarbital concentrations in hair, when plasma concentrations were considered, codeine was apparently incorporated to a 15-fold greater extent than phenobarbital. Because hair pigmentation may be important in drug incorporation, the incorporation of these two drugs was also studied in Long-Evans (LE; produces both black and white hair on the same animal) rats after 40 mg/kg/day of ip drug administration for 5 days. Hair was collected at the same time as the previous experiment. Concentrations of codeine in hair were 44-times greater in pigmented than nonpigmented hair from the same animals. In contrast, hair concentrations of phenobarbital were identical in both pigmented and nonpigmented hair. These data suggest that hair pigmentation greatly affects weak base incorporation but not weak acid incorporation into hair. Because hair concentrations of phenobarbital are not affected by pigmentation, phenobarbital may be an ideal drug to separate out factors other than pigmentation involved in incorporation of drugs into hair.

Animals↗
PubMed1996

Mechanisms for tolerance to methamphetamine effects.

Pretreatment with incremental increases in methamphetamine causes tolerance to serotonergic effects caused by challenging with multiple high doses of methamphetamine (11.5 mg/kg/dose). The brain concentration of methamphetamine following this challenge was reduced in tolerant rats, yet the plasma concentration was elevated. The tolerance was selective for methamphetamine and did not occur when cocaine was used in the pretreatment. The possibility that tolerance affects the distribution of methamphetamine in or out of the brain through an active transport system was examined by combining the transport-blocking drug, probenecid, with a low dose of methamphetamine. The presence of probenecid enhanced methamphetamine-induced serotonergic changes in the hippocampus. The brain concentration of methamphetamine increased in the presence of probenecid; however, a similar increase in the plasma methamphetamine concentration suggests that the effects of probenecid on methamphetamine distribution are not related to the redistribution of methamphetamine that occurs in tolerant animals.

Animals↗
PubMed1996

Quantitative analysis of methadone and two major metabolites in hair by positive chemical ionization ion trap mass spectrometry.

A sensitive and specific method for the quantitative determination of D,L-methadone (MD) and its metabolites, D,L-2-ethyl-1,5-dimethyl-3, 3-diphenylpyrrolinium (EDDP) and D,L-2-ethyl-5-methyl-3, 3-diphenyl-1-pyrroline (EMDP), in hair has been developed. Deuterated internal standards of MD, EMDP, and EDDP were added to 20-mg hair samples and digested overnight at room temperature with 1N sodium hydroxide. Calibration standards containing known concentrations of MD, EMDP, and EDDP dried onto human hair were also digested. Digest solutions were extracted by a liquid-liquid extraction procedure and analyzed with splitless injection on a Finnigan MagnumTM ion trap mass spectrometer. Chromatographic separation was achieved with helium carrier gas on a DB-5MS-30M-0.25-micron capillary column. Positive chemicaionization was used with acetone as the reagent gas. The assay was linear from 0.5 ng/mg (MD and EDDP) or 1.0 ng/mg (EMDP) to 50.0 ng/mg of human hair with correlation coefficients greater than 0.99. Intra-assay and interassay coefficients of variation were determined to be less than 20% for all three analytes at 2.0 and 10.0 ng/mg of hair. Recovery was estimated to be greater than 70% (MD and EDDP) and 53% (EMDP) at 2.0 and 10.0 ng/mg of hair. The method has been applied to the analysis of both human and rat hair. Male long-Evans rats were shaved prior to dosing to obtain their drug-free hair. Animals were then administered 15 mg/kg MD by intraperitoneal injection daily for five days. Fourteen days after the first dose, hair was collected and analyzed for MD, EMDP, and EDDP. The mean plus standard error of the mean (SEM; n = 3) concentrations of MD and EDDP in pigmented hair were 31.1 ng/mg +/- 9.6 ng/mg and 8.6 +/- 2.4 ng/mg, respectively. EMDP was detected in the hair of one of three rats. In another experiment, hair was collected from two human subjects who had received long-term methadone therapy for the treatment of heroin addiction. Subject A received 60 mg of methadone daily for at least six months; subject B received 80 mg of methadone daily for at least six months. The hair concentrations of MD were 10.1 ng/mg and 21.0 ng/mg for Subjects A and B, respectively. The hair concentrations of EDDP were 0.5 ng/mg and 2.6 ng/mg for Subjects A and B, respectively. EMDP was not detected in the hair of these two subjects. This method is being used to evaluate the incorporation of MD, EMDP, and EDDP in human and rat hair in dose-response studies.

Animals↗
PubMed1996

Incorporation of codeine and metabolites into hair. Role of pigmentation.

Xenobiotics circulating in the blood may become incorporated into growing hair. Melanin has affinity for many pharmacologically unrelated drugs and is responsible for the pigmentation in hair. To assess the role of pigmentation in the incorporation of drugs into hair, the distribution of codeine and its metabolites was studied in Sprague-Dawley (SD; white nonpigmented hair), Dark Agouti (DA; brown pigmented hair), and hooded Long-Evans (LE; both black pigmented and white nonpigmented hair) rats. Codeine was administered at a dose of 40 mg/kg/day i.p. for 5 days. Fourteen days after beginning the dosing protocol, hair was collected and analyzed for codeine, and its metabolite, morphine, by positive-ion chemical ionization GC/ion-trap MS. The plasma pharmacokinetics for codeine and morphine were also determined after a single 40 mg/kg injection (equivalent to first dose in 5-day dosing protocol) in all three strains of rats. Hair and plasma codeine and morphine concentrations were also determined after acid hydrolysis to evaluate the presence of glucuronide metabolites. Codeine concentrations in the hair of SD, DA, and pigmented LE hair were 0.98 +/- 0.10, 5.99 +/- 1.24, and 111.93 +/- 18.69 ng/mg hair, respectively; morphine concentrations were 0.34 +/- 0.04, 0.51 +/- 0.11, and 14.46 +/- 1.81 ng/mg hair, respectively; morphine glucuronide concentrations were 0.67 +/- 0.08, 1.04 +/- 0.37, and 13.80 +/- 3.60 ng/mg hair, respectively. Studies examining the in vitro binding of [3H] codeine and [3H]morphine to hair demonstrated both specific and nonspecific binding sites for codeine and morphine. Pigmented hair from LE rats possessed the greatest number of binding sites, white hair from SD rats contained the least, and brown hair from DA rats was intermediate. A time course study of codeine and its metabolites showed pigment-mediated differences in incorporation of codeine and metabolites within a few hours of drug administration. These data indicate that pigmented hair possesses a greater capacity to bind and incorporate codeine and its metabolites than does nonpigmented hair. Interpretation of hair concentrations of drugs should involve consideration of hair pigmentation.

Animals↗
PubMed1996

Dose-related distribution of codeine and its metabolites into rat hair.

Drugs and endogenous compounds may be incorporated into the matrix of a growing hair shaft. However, the relationship between incorporation and dose or time course of plasma concentrations is poorly defined. The purpose of this study was to compare plasma and hair concentrations of codeine and its metabolites after various doses of codeine. Male Sprague-Dawley rats had a 1" x 1" square shaved from their backs. Codeine was administered by intraperitoneal injection (10, 20, 40, or 60 mg/kg/day) daily for 5 days. Fourteen days after beginning drug administration, the original patch was reshaved and newly grown hair was analyzed for codeine and morphine using GC/MS. The mean concentrations of codeine in hair for the 10, 20, 40, and 60 mg/kg/day groups were 0.29, 0.57, 0.96, and 1.93 ng/mg hair, respectively, and the concentrations of morphine were 0.15, 0.28, 0.49, and 0.79 ng/mg hair, respectively. The plasma concentration time courses for codeine and morphine were determined after single doses of either 20 or 40 mg/kg. Peak plasma codeine concentrations for the 20 and 40 mg/kg groups were 1,441 and 2,452 ng/ml plasma, respectively, and the areas under the plasma concentration vs. time curve were 699 and 1,581 ng-hr/ml, respectively. Morphine glucuronide, but not codeine glucuronide, was measured in the hair of rats administered codeine. Codeine was also administered to rats by constant intravenous infusion (40 mg/kg/day for 5 days). The concentration of codeine in rat hair after this route of administration was 2.92 +/- 0.72 ng/mg hair. Codeine and morphine are incorporated into rat hair in a dose-proportional fashion. Morphine glucuronide can be found in rat hair after codeine administration. The codeine concentration in hair is the same whether the drug is administered by constant intravenous infusion or daily intraperitoneal injections if the areas under the plasma concentration vs. time curve values are considered.

Animals↗
PubMed1995

Distribution of codeine and morphine into rat hair after long-term daily dosing with codeine.

Hair analysis for drugs of abuse provides a possible long-term measure of drug use not possible with urinalysis. Many drugs and their metabolites have been detected in hair; however, the factors influencing the incorporation of chemicals into hair are poorly understood. An animal model for chemical uptake into hair utilizing controlled drug administration was developed to ascertain if increasing doses of codeine are reflected in the concentrations of codeine and its metabolites found in hair. Male Sprague-Dawley rats were administered codeine at 5, 10, or 20 mg/kg (intraperitoneally; n = 6) daily for 21 days. At various times during and after the dosing protocol, approximately 50 mg of hair was shaved from a different area of the animals' backs and analyzed for codeine and morphine concentrations by ion-trap gas chromatography-mass spectrometry. Peak hair codeine concentrations for the 5-, 10-, and 20-mg/kg groups occurred 20 days after beginning the dosing protocol and were 0.57 +/- 0.13, 0.80 +/- 0.10, and 1.95 +/- 0.35 ng/mg hair, respectively. Morphine peak concentrations occurred at the same time and were 1.08 +/- 0.28, 1.21 +/- 0.09, and 2.10 +/- 0.26 ng/mg hair for the 5-, 10-, and 20-mg/kg groups, respectively. Long-term dosing in the rat resulted in similar or greater hair concentrations of morphine (metabolite) than codeine. The plasma pharmacokinetics of codeine and morphine were also obtained after a single, intraperitoneal codeine administration of 20 mg/kg. An experiment involving washing the rat hair with methanol or phosphate buffer (pH 9.0) did not reduce the concentration of codeine or morphine measured in hair as compared with nonwashed control hair. Data obtained in this study indicate that after controlled administration the incorporation of codeine and its metabolite, morphine, into rat hair occurs in a distinct dose-proportional manner.

Animals↗
PubMed1994

Differential effects of antipsychotic and psychotomimetic drugs on neurotensin systems of discrete extrapyramidal and limbic regions.

The effects of two antipsychotic (dopamine antagonist) drugs, haloperidol and clozapine, and of two psychotomimetic (dopamine-releasing) drugs, methamphetamine and cocaine, on neurotensin (NT) concentrations in discrete regions of the caudate nucleus, nucleus accumbens and globus pallidus were examined. Multiple administrations of haloperidol (HA, 1 mg/kg), clozapine (20 mg/kg), methamphetamine (METH, 10 mg/kg) or cocaine (30 mg/kg) increased NT-like immunoreactivity (NTLI) in the whole striatum (caudate nucleus plus globus pallidus). The effects of combined HA and METH treatment on striatal NTLI were additive. In contrast, the effects of clozapine plus METH were not different from those caused by either drug alone. The caudate nucleus, nucleus accumbens and globus pallidus were dissected into nine areas based on anterior-posterior and medial-lateral position. Across the caudate areas, some differences in NTLI concentrations occurred when cocaine- and METH-treated groups were compared, even though whole striata in these groups did not differ significantly. The effects of the antipsychotic drugs in discrete caudate regions, alone or in combination with METH, confirmed the observations in the whole striata, although significant regional differences existed. There were also differential regional effects in the nucleus accumbens. Drug-induced changes in the NTLI content of the anterior nucleus accumbens were similar to those observed in the whole striatum, whereas NTLI changes in the posterior region of this structure often were opposite. Finally, NTLI concentrations in the globus pallidus were increased by Ha or METH treatment, but were not affected by clozapine or cocaine treatment. These findings suggest that NT systems throughout the entire striatum and nucleus accumbens do not respond in a homogeneous manner to these drugs which stimulate or block dopamine activity. The regional differences in NT responses may reflect different dopamine neurons which are affected differentially by dopamine-altering drugs.

Animals↗
PubMed1993

Blockade of tachykinin NK1 receptors by CP-96345 enhances dopamine release and the striatal dopamine effects of methamphetamine in rats.

The nonpeptide, tachykinin NK1 receptor antagonist, CP-96345, permits the study of the physiological role of extrapyramidal substance P systems. Using microdialysis, we observed that locally applied CP-96345 (200 nM) caused a significant increase in dopamine release in the striatum as well as substantially enhancing striatal dopamine release caused by a low dose of methamphetamine (0.5 mg/kg s.c.). In addition, multiple systemic administrations of CP-96345 almost doubled the dopamine-mediated responses of the striatal neurotensin and dynorphin systems to high doses of methamphetamine (10 mg/kg/dose s.c.). Our findings suggest that the physiological role of substance P released in the striatum is to decrease the activity of the nigrostriatal dopamine pathway.

Animals↗
PubMed1991

Divergence of muscle and liver fructose 2,6-diphosphate in fasted exercising rats.

Previous studies demonstrate that nonexercising muscle may serve as a source of lactate for hepatic gluconeogenesis during long-term exercise. The concentration of fructose 2,6-diphosphate (F-2,6-P2), a signal molecule that accelerates glycolysis, was examined in liver and muscles of fed and fasted resting rats and in fasted rats run for 5, 15, or 30 min at 21 m/min (15% grade). Liver F-2,6-P2 decreased in response to fasting and exercise. White quadriceps (composed predominantly of type IIb fibers) F-2,6-P2 increased from 2.2 +/- 0.1 to 4.5 +/- 0.4 pmol/mg in the fasted rats in response to 30 min of treadmill running. No increase was observed in the red region of the quadriceps (composed of type IIa fibers). The fasted rats also exhibited a threefold increase in glucose 1,6-diphosphate (G-1,6-P2) in the white quadriceps after 30 min of exercise, whereas no significant changes were observed in the red quadriceps or in liver. The increases in F-2,6-P2 and G-1,6-P2 may be important in accelerating glycolysis and enhancing lactate production in muscles that are not glycogen depleted during long-term exercise.

Animals↗