Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples. Copyright (C) 2004 John Wiley Sons, Ltd.

Ultrafast dynamics of dye molecules in solution as a function of temperature

Femtosecond time-resolved studies using fluorescence depletion spectroscopy were performed on Rhodamine 700 in acetone solution and on Oxazine 750 in acetone and formamide solutions at different temperatures. The experimental curves that include both fast and slow processes have been fitted using a biexponential function. Time constants of the fast process, which corresponds to the intramolecular vibrational redistribution (IVR) of solute molecules, range from 300 to 420 fs and increase linearly as the temperature of the environment decreases. The difference of the average vibrational energy of solute molecules in the ground state at different temperatures is a possible reason that induces this IVR time-constant temperature dependence. However, the time constants of the slow process, which corresponds to the energy transfer from vibrational hot solute molecules to the surroundings occurred on a time scale of 1-50 ps, changed dramatically at lower temperature, nonlinearly increasing with the decrease of temperature. Because of the C-H...O hydrogen-bond between acetone molecules, it is more reasonable that acetone molecules start to be associated, which can influence the energy transfer between dye molecules and acetone molecules efficiently, even at temperatures far over the freezing point.

Study of competitive binding of enantiomers to protein by affinity capillary electrochromatography

Affinity capillary electrochromatography (CEC) with zonal elution method was used to probe the competitive interactions of enantiomers with protein. In this approach, a known concentration of a competing agent is continuously applied to a CEC column with bovine serum albumin (BSA) physically adsorbed on SAX packing while injections of a small amount of analyte are made. The binding sites of solutes on the BSA molecule were determined by the changes in the retention factors of the solutes resulted from the addition of competitive agent. By using D- or L-tryptophan as competitive agents and D-, L-tryptophan and benzoin enantiomers as injected analytes showed that BSA molecule has a primary site to strongly bind L-tryptophan, but D-tryptophan dose not bind at this site; D- and L-tryptophan share a weak binding site on the BSA molecule. Benzoin enantiomers do not share any binding sites with either D- or L-tryptophan. Non-chiral compounds of trichloroacetic acid and n-hexanoic acid were applied as the competitive agents to study the binding of warfarin enantiomers to BSA, it was observed that trichloroacetic acid and n-hexanoic acid had a same binding site for warfarin enantiomers binding to BSA molecule. (C) 2002 Elsevier Science B.V. All rights reserved.

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site. proved to be the preferential one, being 6.50 x 10(5) 1.94 x 10(6) and 8.94 x 10(5). respectively. In addition. it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions. (C) 2002 Elsevier Science B.V. All rights reserved.

Affinity membrane chromatography for the analysis and purification of proteins

Affinity chromatography is unique among separation methods as it is the only technique that permits the purification of proteins based on biological functions rather than individual physical or chemical properties. The high specificity of affinity chromatography is due to the strong interaction between the ligand and the proteins of interest. Membrane separation allows the processing of a large amount of sample in a relatively short time owing to its structure, which provides a system with rapid reaction kinetics. The integration of membrane and affinity chromatography provides a number of advantages over traditional affinity chromatography with porous-bead packed columns, especially with regard to time and recovery of activity. This review gives detailed descriptions of materials used as membrane substrates, preparation of basic membranes, coupling of affinity ligands to membrane supports, and categories of affinity membrane cartridges. It also summarizes the applications of cellulose/glycidyl methacrylate composite membranes for proteins separation developed in our laboratory. (C) 2001 Elsevier Science B.V. All rights reserved.