Shotgun protein analysis by liquid chromatography-tandem mass spectrometry.

Two-dimensional electrophoresis (2DE) is an excellent technology for the analysis of complex protein mixtures, but it has drawbacks, such as hardly detecting very hydrophobic proteins. Shotgun protein analysis is one of the major technologies used to compensate for the weaknesses of 2DE. In this approach, total proteins are digested as a mixture and the digested peptides are separated by one-dimensional or multidimensional chromatography and introduced into a tandem mass spectrometer.

Cloning, expression, and characterization of male cynomolgus monkey liver aldehyde oxidase.

In this study, we investigated the properties of monkey liver aldehyde oxidase directed toward the clarification of species differences. The aldehyde oxidase preparation purified from male cynomolgus monkey liver cytosol showed a major 150 kDa Coomassie brilliant blue (CBB)-stained band together with a minor 130 kDa band using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both bands were identified as being aldehyde oxidase by a database search of the MS data obtained with nano-liquid chromatography, quardrupole time of flight, mass spectrometry (nano-LC Q/TOF MS).

Chiral inversion of RS-8359: a selective and reversible MAO-A inhibitor via oxido-reduction of keto-alcohol.

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine is a selective and reversible MAO-A inhibitor. The (S)-enantiomer of RS-8359 has been demonstrated to be inverted to the (R)-enantiomer after oral administration to rats. In the current study, we investigated the chiral inversion mechanism and the properties of involved enzymes using rat liver subcellular fractions.

Expression of the theta class GST isozyme, YdfYdf, in low GST dogs.

We have reported the existence of low glutathione S-transferase (GST) dogs whose GST activity to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activity) is quite low, and have also reported significant individual differences in dog liver GST-D activity. The dogs were classified as "low", "middle", or "high" GST dogs based on their GST-D activity. In the present study, in order to investigate the causes of quite low GST-D activity in low GST dogs and the individual differences in dog GST-D activity, glutathione (GSH) conjugation of DCNB was kinetically analyzed.

Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry.

We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry.

Combination of two-dimensional electrophoresis and shotgun peptide sequencing in comparative proteomics.

Two-dimensional electrophoresis (2-DE) and shotgun peptide sequencing are the two major technologies to compare the expression profile of proteins, which is also referred to as comparative proteomics or quantitative proteomics. Although the methodologies, such as difference gel electrophoresis for 2-DE and isotope-coded affinity tags for shotgun peptide sequencing, have made rapid progress, these two approaches have their own strengths and weaknesses. Therefore, the combination of the two methodologies is beneficial for the purpose of better comparative proteomics, especially in comprehensive coverage of the proteome and protein information such as post-translational modifications.

Evaluation of the protein binding ratio of drugs by a micro-scale ultracentrifugation method.

Ultracentrifugation methods have been widely used for the determination of the free fraction of compounds in plasma, especially for lipophilic compounds. To estimate the effect of contaminated proteins in the "protein-free phase" fraction, 200 microL of human plasma was separated into three layers by ultracentrifugation at 436,000g for 140 min with a table-top ultracentrifuge. Twenty microliters of the middle layer was taken as the protein-free fraction.

Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis.

Hydrolysis of synthetic substrate, L-pyroglutamyl p-nitroanilide is catalyzed solely by pyroglutamyl aminopeptidase I in rat liver cytosol.

Pyroglutamyl aminopeptidase I (PAP-I) is a cytosolic cysteine peptidase, which hydrolytically removes the L-pyroglutamate residue from the amino terminus of endogenous proteins and peptides. L-Pyroglutamyl p-nitroanilide serves as the synthetic substrate of this enzyme, while there is a possibility of other hydrolases being involved in the hydrolysis of this xenobiotic substrate. We cloned a full-length cDNA encoding rat PAP-I from a rat liver cDNA library and expressed this cDNA in Escherichia coli to obtain a recombinant PAP-I as a single protein.