Utility of polyclonal antibodies targeted toward unique tryptic peptides in the proteomic analysis of cytochrome P450 isozymes.

P450s are key enzymes responsible for biotransformation of numerous endogenous and exogenous compounds and are located in almost every tissue. This superfamily is the largest group of enzymes (>6000) that share a high degree of similarity in protein sequence. The human genome contains 57 CYP genes and 58 pseudogenes. A major gap exists in our knowledge about differences in CYP expression on a protein level. DNA and mRNA information are not sufficient because transcription and particularly translation events are not necessarily correlated with levels of expressed proteins. The data reported in this study complete the framework of an integrated proteomic method for the simultaneous qualitative and quantitative analysis of CYP isozyme composition using MALDI-TOF-MS and immunochemistry that has been developed in our laboratory over the last several years (Alterman et al., 2005a,b) and is based on the fact that each P450 isozyme possesses unique tryptic peptide(s) (UTP) that could be used for differential analysis of human CYP expression. Here we demonstrate that three different immunochemical techniques (ELISA, Western blot, and peptide affinity enrichment on magnetic beads with attached antibodies) have potential to be incorporated in an integrated proteomic method combining mass spectrometry and immunochemistry. Fundamentally, this approach is based on the measurement of the same chemical entity (isozyme-specific UTP) in the tryptic digest by two orthogonal analytical techniques, mass spectrometry and immunochemistry. The application of this approach is illustrated with two human CYP isozymes--CYP1A2 and CYP2E1.

High-throughput screening for Hsp90 ATPase inhibitors.

Recently, we reported a useful assay for the determination of yeast Hsp90 ATPase activity. Using this assay, high-throughput screening of approximately 10,000 compounds was performed to determine the feasibility of this assay on large scale. Results from high-throughput screening indicated that the assay was reproducible (av Z-factor = 0.80) and identified 0.57% of the compounds as Hsp90 inhibitors that exhibited IC50s less than 20 microM. The structures of several of these inhibitory scaffolds are reported along with their IC50 values.

Protein interactions with subcutaneously implanted biosensors.

Biofouling of in vivo glucose sensors has been indicated as the primary reason for sensitivity losses observed during the first 24 h after implant [Wisniewski N, Moussy F, Reichert WM. Characterization of implantable biosensor membrane biofouling. Fresen J Anal Chem 2000; 366(6-7): 611-621]. Identification of the biomolecules that contribute to these sensitivity perturbations is the primary objective of the research presented. Active needle-type glucose sensors were implanted in Sprague-Dawley rats for 24h, and then a proteomics approach was used to identify the substances absorbed to the sensors. MALDI-TOF mass spectrometry was the primary tool utilized to identify the biomolecules in sensor leachate samples and species absorbed directly on sensor membranes excised from explanted in vivo sensors. Not surprisingly serum albumin was identified as the primary biomolecule present, however, predominantly as endogenous fragments of the protein. In addition, several other biomolecule fragments, mainly less than 15 kD, were identified. Based on these findings, it is concluded that fragments of larger biomolecules infiltrate the sensor membranes causing diminished glucose diffusivity, thus decreasing in vivo sensitivity.

Development and optimization of a useful assay for determining Hsp90's inherent ATPase activity.

The Hsp90 molecular chaperone is responsible for the conformational maturation of nascent polypeptides and the rematuration of denatured proteins. Inhibition of Hsp90 represents a promising approach towards the treatment of cancer because numerous signaling cascades can be simultaneously targeted by disruption of the Hsp90-mediated process. Hsp90's ATPase activity is essential to the Hsp90-mediated protein folding process, consequently, a coupled assay was developed and optimized for determination of Hsp90's inherent ATPase activity. Using maltose phosphorylase, glucose oxidase, and horseradish peroxidase as components of this assay, a highly reproducible assay with a Z-factor of 0.87 has been produced.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based amino acid analysis.

Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids. This method does not require any amino acid modification, derivatization, or chromatographic separation. The data acquisition time is decreased to several seconds for a single sample. No significant ion suppression effects were observed with the developed sample deposition technique, and the method was found to be reproducible. Linear responses between the amino acid concentration and the peak intensities ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 microM, and correlation coefficients were between 0.983 (for arginine) and 0.999 (for phenylalanine). Limits of quantitation were between 0.03 microM (for arginine) and 3.7 microM (for histidine and homocysteine). This method was applicable to the mixtures of free amino acids as well as to HCl hydrolysates of proteins. Furthermore, we have shown that this method can be applied to other biologically important low-molecular weight compounds such as glucose.