A comprehensive platform for quality control of botanical drugs (PhytomicsQC): a case study of Huangqin Tang (HQT) and PHY906.

BACKGROUND: Establishing botanical extracts as globally-accepted polychemical medicines and a new paradigm for disease treatment, requires the development of high-level quality control metrics. Based on comprehensive chemical and biological fingerprints correlated with pharmacology, we propose a general approach called PhytomicsQC to botanical quality control. METHODS: Incorporating the state-of-the-art analytical methodologies, PhytomicsQC was employed in this study and included the use of liquid chromatography/mass spectrometry (LC/MS) for chemical characterization and chemical fingerprinting, differential cellular gene expression for bioresponse fingerprinting and animal pharmacology for in vivo validation. A statistical pattern comparison method, Phytomics Similarity Index (PSI), based on intensities and intensity ratios, was used to determine the similarity of the chemical and bioresponse fingerprints among different manufactured batches. RESULTS: Eighteen batch samples of Huangqin Tang (HQT) and its pharmaceutical grade version (PHY906) were analyzed using the PhytomicsQC platform analysis. Comparative analysis of the batch samples with a clinically tested standardized batch obtained values of PSI similarity between 0.67 and 0.99. CONCLUSION: With rigorous quality control using analytically sensitive and comprehensive chemical and biological fingerprinting, botanical formulations manufactured under standardized manufacturing protocols can produce highly consistent batches of products.

Structural proteomics of macromolecular assemblies using oxidative footprinting and mass spectrometry.

Understanding the composition, structure and dynamics of macromolecules and their assemblies is at the forefront of biological science today. Hydroxyl-radical-mediated protein footprinting using mass spectrometry can define macromolecular structure, macromolecular assembly and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side-chain groups with covalent-modification reagents. Subsequent to oxidation by reactive oxygen species, proteins are digested by specific proteases to generate peptides for analysis by mass spectrometry. Accurate measurements of side-chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side-chain sites within the macromolecular probes are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes.

Structure and dynamics of the actin filament.

The structures of filamentous Mg-ATP-actin (F actin) in the presence and absence of KCl have been mapped with hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry (MS) analysis revealed 52 reactive side-chain sites from 27 distinct peptides within actin. The reactivities of these probe sites with *OH in the F-actin states are compared with those of Mg-ATP-G-actin (monomers) analyzed previously [Guan, J.-Q. et al. (2003) Biochemistry 42, 11992-12000]. Filament-dependent protection within subdomains 2, 3, and 4 and at the C terminus is consistent with longitudinal contacts of monomers within the filament helical structure as predicted by the Holmes model. In the absence of KCl, the extent of filament-dependent protection rarely reached 3-fold, consistent with a highly dynamic filament characterized by relatively weak interactions between actin protomers. However, in the presence of KCl, the extents of protection are significantly increased, consistent with a well-ordered, more tightly packed filament structure. Filament-dependent enhancements of reactivity not predicted by the Holmes model are seen for a peptide that overlaps the "hydrophobic plug" (H-plug) region and for a peptide that forms contacts with the polyphosphate moiety of the bound nucleotide. Overall, these data are both consistent with and complementary to a recent deuterium-exchange MS study of filamentous actin [Chik, J. K., and Schriemer, D.C. (2003) J. Mol. Biol. 334, 373-385], which also did not detect any burial of the H plug upon formation of filaments.

Synchrotron radiolysis and mass spectrometry: a new approach to research on the actin cytoskeleton.

Hydroxyl radicals generated from millisecond exposure of aqueous solutions to synchrotron X-rays react with proteins to yield stable oxidative modifications of solvent-accessible amino acid side chains. Following proteolysis, HPLC/MS analysis is performed to quantitate the side chain reactivities, and MS/MS analysis is used to identify the modification site(s). Side chain reactivity is shown to be correlated with solvent accessibility; thus the method provides detailed site-specific information about protein structure. The application of these techniques to the study of the actin cytoskeleton is described in detail, including defining the binding sites of monomeric actin with gelsolin segment-1, the actin monomer binding surface on cofilin, the divalent cation-dependent structure changes of monomeric actin, and the conformational changes associated with actin filamentous assembly.

Structural reorganization of proteins revealed by radiolysis and mass spectrometry: G-actin solution structure is divalent cation dependent.

The solution structures of isolated monomeric actins in their Mg(2+)-ATP and Ca(2+)-ATP bound forms and in complexes with gelsolin segment-1 have been probed using hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry analysis of 28 peptides containing 58 distinct reactive probe sites within actin were used to monitor conformational variations linked to divalent cation and gelsolin segment-1 binding. The solvent accessibilities of the probe sites, as measured by footprinting in solution for the Ca(2+)-G-actin and Mg(2+)-G-actin complexes with gelsolin segment-1, were consistent with available crystallographic data. This included a specific protection at the contact interface between the partners, as revealed by reduced reactivity of peptide 337-359 in the complex. Aside from the specific protection indicated previously, the oxidation rates for the reactive residues of the isolated Ca(2+)-G-actin were similar to those of the actin gelsolin segment-1 complexes; however, the reactivity of numerous residues in the isolated Mg(2+)-G-actin form was significantly reduced. Specifically, Mg(2+)-G-actin has a set of protected sites relative to Ca(2+)-G-actin that suggest a structural reorganization in subdomains 4 and 2 and a C-terminus more closely packed onto subdomain 1. These conformational variations for isolated Mg(2+)-G-actin provide a structural basis for its greater tendency to polymerize into filaments as compared to Ca(2+)-G-actin.

Structural reorganization of the transferrin C-lobe and transferrin receptor upon complex formation: the C-lobe binds to the receptor helical domain.

Human transferrin, a bilobal protein, with each lobe bearing a single iron-binding site, functions to transport iron into cells. While the N-terminal lobe alone does not measurably bind cellular transferrin receptors or serve as an iron donor for cells, the C-lobe is capable of both functions. We used hydroxyl radical-mediated protein footprinting and mass spectrometry to reveal the conformational changes that occur upon complex formation for the human transferrin C-lobe (residues 334-679) bound to the ectodomain of human transferrin receptor 1 (residues 121-760). Oxidation rates for proteolytic peptides in the C-lobe, the receptor, and their complex have been measured by mass spectrometry; upon formation of the complex, a dramatic decrease in modification rates, indicating protection of specific side chain groups, can be seen in C-lobe sequences corresponding to residues 381-401, 415-433, and 457-470. Peptide sequences experiencing modification rate decreases in the transferrin receptor upon C-lobe binding include residues 232-240, 365-371, 496-508, 580 and 581, 614-623, 634-646, 647-681, and 733-760. In addition, several peptides in the receptor exhibit enhancements in the rate of modification consistent with allosteric effects of complex formation. Using tandem mass spectrometry, the sites of modification with altered reactivity in the complex include Met382, Met389, Trp460, Met464, and Phe427 in the C-lobe and Tyr503, Pro581, Tyr611, Leu619, Met635, Phe650, Trp740, Trp754, and Phe760 within the transferrin receptor. Using available genetic, biochemical, and structural data, we confirm that the conserved RGD sequence (residues 646-648) in the helical domain of the transferrin receptor, including residues from Leu619 to Phe650, is a primary binding site for the transferrin C-lobe.

Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting.

Cofilin is an actin regulatory protein that binds to both monomeric and filamentous actin, and has filament severing activity. Although crystal structures for the monomeric forms of both G-actin and cofilin have been described, the structure of the binary cofilin-G-actin complex is not available. Synchrotron protein footprinting is used to identify specific side chain residues on the cofilin surface that are buried in the formation of the cofilin-G-actin binary complex. Exposure to synchrotron X-rays results in stable oxidative modifications of aromatic, aliphatic, and sulfur-containing side chains, with the rate of modification for a particular residue being dependent on its intrinsic reactivity and solvent accessibility. The rates of modification were monitored for a number of peptides generated by digestion of oxidized cofilin, both in isolation and in its binary complex with G-actin. After binding to G-actin takes place, a significant decrease in modification rates, indicating protection of side chain groups, is seen for cofilin peptides corresponding to residues 4-20, 10-17, 83-96, 91-105, and 106-117. A number of other peptides show no change in reactivity, and are presumed to represent regions distal to the binding site. Tandem mass spectrometry demonstrates that residues Leu 13, Pro 94, Met 99, and Leu 108 and 112 directly participate in the binding interface. These results are generally consistent with, and complementary to, the results of previous site-directed mutagenesis studies and extend our understanding of the G-actin binding surface of cofilin.

Regioselectivity in the Photocycloaddition of 9-Substituted Anthracenes Incorporated within Nafion Membranes.

The photocycloaddition of five 9-substituted anthracenes [AnCH(2)N(+)(CH(3))(3)Br(-) (1), AnCH(2)COO(-)Na(+) (2), AnCH(2)OH (3), AnCOCH(3) (4), and AnCH(3) (5), An = 9-anthryl] incorporated within Nafion membranes has been investigated. While irradiation of the above compounds in homogeneous solutions, and 5 in Nafion also, mainly gave rise to their head-to-tail (h-t) photocyclomers, photoirradiation of the samples of 1-4 incorporated within Nafion membranes almost exclusively resulted in the head-to-head (h-h) photocyclomers. Furthermore, the h-h photocyclomers are significantly more stable in Nafion membranes than in homogeneous solutions. These observations can be explained in terms of the preorientation of the substrate molecules in the inverse micelle-like clusters of Nafion.