Identifying protein stabilizing ligands using GroEL.

Over the past 5 years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (approximately 30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called orphan protein folding diseases. The development of a broad-based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic, and medical professions.

Strategies for folding of affinity tagged proteins using GroEL and osmolytes.

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.

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.

Quantitative analysis of cytochrome p450 isozymes by means of unique isozyme-specific tryptic peptides: a proteomic approach.

A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.

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.