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.

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.

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.

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.

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).

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.

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.

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.