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. Since the shotgun approach is the primary strategy in proteomics besides 2DE, a great number of related methodologies have been developed. In this chapter, we would like to introduce the simplest protocol, in which proteins are digested in solution and the digested peptides are analyzed by one-dimensional reversed-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), as a starting point for shotgun protein analysis.

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). Based on the sequence coverage, the 130 kDa protein was presumed to be deficient in 20-30 kDa mass from the N-terminus. Full male cynomolgus monkey aldehyde oxidase cDNA was cloned and sequenced with the four degenerate primers designed by considering the peptide sequences containing the amino acids specific for monkey aldehyde oxidase. The deduced amino acid sequences had 96% amino acid identity with those of human enzyme. The aldehyde oxidase expressed in Escherichia coli also exhibited two immunoreactive bands on SDS-PAGE/Western blot analysis. Further, the biphasic pattern was observed for Eadie-Hofstee plots of the (S)-enantiospecific 2-oxidation activity of RS-8359 with the expressed and cytosolic monkey liver aldehyde oxidase. The results suggested that two forms of aldehyde oxidase in monkey were the expression products by a single gene. In contrast, the similarly expressed rat aldehyde oxidase showed only one immunoreactive protein and monophasic pattern. The biphasic phenomenon could be caused by the existence of two aldehyde oxidase isoforms or two active sites in a single enzyme or some other reasons. Further studies on the problems of the biphasic pattern and species differences in aldehyde oxidase are needed.

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. The 7-hydroxy function of RS-8359 was oxidized at least by the two different enzymes. The cytosolic enzyme oxidized enantiospecifically the (S)-enantiomer with NADP as a cofactor. On the other hand, the microsomal enzyme catalyzed more preferentially the oxidation of the (S)-enantiomer than the (R)-enantiomer with NAD as a cofactor. With to product enantioselectivity of reduction of the 7-keto derivative, it was found that only the alcohol bearing (R)-configuration was formed by the cytosolic enzyme with NADPH and the microsomal enzyme with NADH at almost equal rate. The reduction rate was much larger than the oxidation rate of 7-hydroxy group. The results suggest that the chiral inversion might occur via an enantioselectivity of consecutive two opposing reactions, oxidation and reduction of keto-alcohol group. In this case, the direction of chiral inversion from the (S)-enantiomer to the (R)-enantiomer is governed by the enantiospecific reduction of intermediate 7-keto group to the alcohol with (R)-configuration. The enzyme responsible for the enantiospecific reduction of the 7-keto group was purified from rat liver cytosolic fractions and identified as 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) via database search of peptide mass data obtained by nano-LC/MS/MS.

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. Moreover, liver cytosolic proteins whose expression levels were significantly lower in low GST dogs than in high GST dogs were identified by two-dimensional difference gel electrophoresis (2D-DIGE) and LC tandem mass spectrometry (LC/MS/MS). Interestingly, Vmax values for this reaction well reflected their GST-D activities in all groups, i.e. they were 3.8, 80.6, and 169.2 nmol/min/mg protein in the low, middle, and high GST dogs, respectively. However, Km values were almost the same (260.0-283.7 microM) among these groups. These suggest that GSH conjugation of DCNB should be catalyzed by the same enzyme in all the dogs, and individual differences in the GST-D activity should be the result of individual differences in the expression level of the GST isozyme, which catalyzes conjugation of DCNB. In 2D-DIGE, the expression levels of the two protein spots were significantly lower in the low GST dogs than in the high GST dogs. Positive good correlation (r > 0.800) was observed between GST-D activity and expression levels of these two protein spots. Moreover, expression levels were quite low in low GST dogs. These two proteins were both identified as the theta class GST isozyme, YdfYdf, which specifically catalyzes GSH conjugation of DCNB in dog livers. In the present study, we present two novel findings based on an enzyme kinetic study and protein-expression analysis: (1) GSTYdfYdf is expressed at quite a low level in the liver of low GST dogs, and (2) individual differences in dog liver GST-D activity would be due to individual differences in the expression level of GSTYdfYdf. Considering these findings, low GST dogs might have high susceptibility, including an unexpected toxicity at abnormal exposure to chemicals metabolized by GSTYdfYdf.

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. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.

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. Major contaminated proteins were analyzed by liquid chromatography/electrospray ionization/tandem mass spectrometry (LC/ESI/MS/MS) and identified as albumin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, apolipoprotein E, and apolipoprotein A-1. alpha-1-acid glycoprotein was not detected. Contamination of albumin was 0.13% of that in plasma. Simulation analysis demonstrated that at an actual free fraction of 1% (protein binding ratio of 99%), the extent of overestimation of free fraction was just 13% and the apparent free fraction was 1.13%. Human plasma protein binding ratios of 10 drugs estimated by this method correlated well with reported values determined by other methods, such as ultrafiltration and equilibrium dialysis, with a correlation factor of 0.98 and a slope of 0.99. Collectively, our results indicate the reliability of this micro-scale ultracentrifugation technique for the evaluation of the protein binding of drugs despite a little contamination of albumin.

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. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.

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. The cDNA encoded a sequence of 209 amino acids with a calculated molecular weight of 22913 Da. The homology of the deduced amino acid sequence of rat PAP-I was 98.6 and 94.3% to mouse and human PAP-Is, respectively. The biochemical properties of the recombinant rat PAP-I were almost identical to those of the recombinant mouse and human PAP-Is and the purified rat liver cytosolic PAP-I in terms of the molecular weight, subunit structure, affinity to the substrate, inhibitor profile and pH optimum. Immunoblot analysis using an antibody raised against recombinant rat PAP-I showed that rat PAP-I is present almost exclusively in the cytosolic fraction of the rat liver. Moreover, the hydrolyzing activity for L-pyroglutamyl p-nitroanilide in rat liver cytosolic fraction was completely inhibited by the antibody, strongly suggesting that this xenobiotic substrate is hydrolyzed solely by PAP-I.