F-PCS measurements on proteins in live mammalian cells.

Pseudocontact shift (PCS) contains rich structural information of proteins in structural and chemical biology. F-PCS is determined in live mammalian cells dual labelling of the target protein with a paramagnetic tag and a F-tag, which is achieved by varied reactivity of solvent exposed cysteines in selection of different types of tags. About 0.

Simultaneous Quantification of Biothiols and Deciphering Diverse GSH Stability in Different Live Cells by F-Tag.

GSH, Cys, Hcy, and HS are important biothiols and play important roles in the living systems. Quantitative and simultaneous determination of these biothiols under physiological conditions is still a challenge. Herein, we developed an effective F-reactive tag that readily interacts with these four biothiols for the generation of stable thioether products that have distinguishable F-chemical shifts.

Simultaneous identification and quantification of amino acids in biofluids by reactive F-tags.

A robust method to identify and quantify amino acids close to physiological conditions by 1D F NMR was established. Each F-derivatized amino acid has its characteristic chemical-shift profile that is readily identified in the mixture of amino acids or in biofluids including fetal bovine serum and cell lysates. The method shows great potential in metabolomics and biochemical analysis.

Dynamic Exchange of the Metal Chelating Moiety: A Key Factor in Determining the Rigidity of Protein-Tag Conjugates in Paramagnetic NMR.

Site-specific labeling of proteins with a paramagnetic tag is an efficient way to provide atomic-resolution information about the dynamics, interactions, and structures of the proteins and protein-ligand complexes. The paramagnetic effects manifested in NMR spectroscopy generally contain paramagnetic relaxation enhancement, pseudocontact shifts (PCSs), and residual dipolar coupling (RDC), and these effects correlate closely with the flexibility of protein-tag conjugates. The rigidity of the paramagnetic tag is greatly important in decoding the structural details of macromolecular complexes, because paramagnetic averaging reduces the PCSs and RDCs.

In-cell destabilization of a homodimeric protein complex detected by DEER spectroscopy.

The complexity of the cellular medium can affect proteins' properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region.

In-Cell EPR Distance Measurements on Ubiquitin Labeled with a Rigid PyMTA-Gd(III) Tag.

Double electron-electron resonance (DEER) measures distances between spin labels attached at well-defined sites in a protein and thus has the potential to report on conformational states of proteins in cells. In this work, we evaluate the suitability of the small and rigid 4PS-PyMTA-Gd(III) spin label for in-cell distance measurements. Three ubiquitin double mutants were labeled with 4PS-PyMTA-Gd(III) and delivered into human HeLa cells by electroporation (EP) and hypotonic swelling (HS).

Advances in separation science applied to metabonomics.

Metabonomics focuses on metabolite profile changes in diverse living systems caused by a biological perturbation. These metabolite signatures can be achieved with techniques such as gas chromatography, high-performance liquid chromatography (ultra-high-performance/pressure liquid chromatography and capHPLC), capillary electrophoresis, and capillary electrochromatography normally hyphenated with MS. In this review we present the latest developments of the abovementioned techniques applied in the field of metabonomics, with applications covering phytochemistry, toxicology and clinical research.