Deriving statistical models for predicting peptide tandem MS product ion intensities.

Improved search algorithms and scoring functions are required before the identification of peptide tandem MS data can be considered to be fully reliable and automatable. The development of models that can accurately predict product ion spectra from a peptide sequence would certainly help achieve this goal, but this firstly requires a better understanding of the process of fragmentation of peptides in the gas-phase. We summarize recent developments in this area and show that the prediction of product ion spectra is feasible and should improve the identification of peptide tandem MS data, especially for peptides that currently give low or insignificant scores with current search algorithms.

An improved procedure for the isolation and purification of nicotinic acetylcholine receptor from Torpedo fuscomaculata electric organ.

The nicotinic acetylcholinergic receptor has been isolated and purified from extracts of the electric organ of the fish Torpedo fuscomaculata. The isolation procedure involves (a) a series of purification steps including preparation of membrane fragments, extraction of receptors with non-ionic detergents and chromatofocusing; (b) a novel fluorimetric titration assay. The purified receptor is isolated following a 9-fold purification with an overall yield of 12% and a specific activity of 4027 nM.g-1. Gel electrophoresis in the presence of sodium dodecylsulphate produced only one major band with molecular weight of 44,600 associated with the alpha-subunit. A comparison is made with other established procedures. Affinity chromatography on cobratoxin CNBr-Sepharose CL4B produced a 6.8-fold purification, 5% yield and 2900 nM.g-1 specific activity, while in ion-exchange chromatography on DEAE Sepharose 6B gave a 4.7-fold purification, 3% yield and specific activity of 1988 nM.g-1.

Protein-ligand interactions: interaction of nitrosamines with nicotinic acetylcholine receptor.

Fluorimetry and spectrophotometry have been used to study the binding of dimethyl, dipropyl, dibutyl and diphenylnitrosamine to nicotinic acetylcholine receptor isolated, and purified, from Torpedo fuscomaculata. Scatchard analysis indicates that all four ligands are true agonists of the receptor exhibiting positive cooperative binding with the existence of more than one class of binding site. The number of binding sites for the nitrosamines approximates 2. Diphenylnitrosamine binds to the receptor more tightly at low concentrations (Kd1 = 1.3 microM) than the aliphatic nitrosamine (Kd1 = 8-12 microM). Yet at high concentrations all nitrosamines behaved with similar Kd values (27-38 microM).