Birendra N. Pramanik

ADP Is the Cognate Ligand for the Orphan G Protein-coupled Receptor SP1999

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to Gαi have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a Gαi-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca2+ mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric Gα subunits (Gαq/i). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC50 of 60 nm. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5′-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5′-O-(thiotriphosphate). Thus, SP1999 is a novel, Gαi-linked receptor for ADP.

Microwave‐enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes

Accelerated proteolytic cleavage of proteins under controlled microwave irradiation has been achieved. Selective peptide fragmentation by endoproteases trypsin or lysine C led to smaller peptides that were analyzed by matrix‐assisted laser desorption ionization (MALDI) or liquid chromatography‐electrospray ionization (LC‐ESI) techniques. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon α‐2b. Most important, using this novel approach digestion of proteins occurs in minutes, in contrast to the hours required by conventional methods.

Electrospray ionization mass spectrometry for the study of non-covalent complexes : an emerging technology

The detection of non-covalent complexes in the mass range 19,000-34,000 Da, using electrospray ionization mass spectrometry (ESI-MS), is reviewed. The examples discussed include (1) a protein-ligand interaction (ras-GDP), (2) an inhibitor-protein-ligand interaction (SCH 54292/SCH 54341-ras-GDP), (3) a protein-protein interaction (gamma-IFN homodimer) and (4) a protein-metal complex [HCV (1-181)-Zn]. In each case, the ESI-MS method is capable of releasing the intact non-covalent complex from its native solution state into the gas phase in the form of multiply-charge ions. The molecular masses of these complexes were determined with a mass accuracy of better than 0.01%, which is far superior to the traditional methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation chromatography. The method provides the researcher with a quick, reliable and reproducible method for probing difficult biological problems. The key to success in the study of non-covalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions; special care must be taken with the source orifice potential and the solution pH and organic co-solvents must be avoided. This paper also illustrates the usefulness of ESI-MS for addressing biological problems leading to the discovery of new therapeutics; the approach involves the rapid screening of potential drug candidates, such as weakly bound inhibitors.

Ras oncoprotein inhibitors: The discovery of potent, ras nucleotide exchange inhibitors and the structural determination of a drug-protein complex

The nucleotide exchange process is one of the key activation steps regulating the ras protein. This report describes the development of potent, non-nucleotide, small organic inhibitors of the ras nucleotide exchange process. These inhibitors bind to the ras protein in a previously unidentified binding pocket, without displacing bound nucleotide. This report also describes the development and use of mass spectrometry, NMR spectroscopy and molecular modeling techniques to elucidate the structure of a drug-protein complex, and aid in designing new ras inhibitor targets.

Application of LC/MS to proteomics studies: current status and future prospects.

Liquid chromatography/mass spectrometry (LC/MS) has become a powerful technology in proteomics studies in drug discovery, including target protein characterization and discovery of biomarkers. This review article will describe current LC/MS approaches in protein characterization, including a bottom-up method for protein identification and quantitative proteomics. We will discuss the investigation of protein post-translational modifications such as glycosylation (glycoproteomics) and phosphorylation (phosphoproteomics) using LC/MS. Future trends in LC/MS with respect to proteomics studies will also be illustrated.

Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions.

A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-γ dimer] and protein-ligand [ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-γ dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-γ dimer is the use of an alkaline solution (pH ≈ 9) for sample preparation and for mass spectrornetry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-γ dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-γ dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.

LC-MS for protein characterization: current capabilities and future trends

With advances in ionization methods and instrumentation, liquid chromatography (LC)/mass spectrometry (MS) has become a powerful technology for protein characterization. This review article will describe the general approaches on LC-MS analysis in protein characterization, including bottom-up and top-down strategies. Discussions will be given on characterization of recombinant proteins, and post-translational and protein modifications such as disulfide bonds, glycosylation and phosphorylation using LC-MS. New research directions in this area will also be presented to illustrate future prospects of LC-MS in protein characterization, including application to proteomics.

Structural characterization of the oxidative degradation products of an antifungal agent SCH 56592 by LC-NMR and LC-MS.

LC-NMR and LC-MS were used to characterize the structures of four major degradation products of SCH 56592, an antifungal drug candidate in clinical trials. These compounds were formed under stress conditions in which the bulk drug substance was heated in air at 150 degrees C for 12 days, and were separated from SCH 56592 as a mixture using a semi-preparative HPLC method. The data from LC-NMR, LC-ESI-MS (electrospray ionization mass spectrometry) and LC-ESI-MS/MS indicate that the oxidation occurred at the piperazine ring in the center of the drug molecule. The structures of the degradation products were determined from the 1H NMR spectra obtained via LC-NMR, which were supported by LC-ESI-MS and LC-ESI-MS/MS analyses. A novel degradation pathway of SCH 56592 was proposed based on these characterized structures.

Solution- and solid-phase synthesis of enantiomerically pure spiro oxindoles

A convenient synthesis of enantiomerically pure oxindoles using a three component reaction involving 1:3 dipolar cycloaddition reaction has been achieved using solution and solid phase chemistry.

Isolation and characterization of a monomethioninesulfoxide variant of interferon α-2b

AbstractPurpose. To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon α-2b. Methods. The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. Results. The rHuIFN α-2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN α-2b molecule. The far-UV CD spectra showed a slight loss of α-helical content and an increase in the β-sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon α-2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. Conclusions. A monomethioninesulfoxide rHuIFN α-2b variant was found to be present in the rHuIFN α-2b bulk drug substance in solution. The Met111 residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met111 residue did not seem to have a detectable effect on the biological activity of the molecule.