Generic multicriteria approach to determine the best precipitation agent for removal of biomacromolecules prior to non-targeted metabolic analysis.

The removal of biomacromolecules from biofluids decreases the sample complexity and lower electrospray suppression effects. Furthermore, it can increase the analysis sensitivity, precision, and selectivity. Often removal approaches evaluate the model based on a single criterion, like protein removed or response of one of few specific metabolites. In this study, we used a multicriteria approach to test the effect of using the solvents methanol and acetonitrile (organic solvent precipitation), trichloroacetic acid (acidic precipitation) and ammonium sulphate (salting out) to remove biomacromolecules from a downstream recovery process from a bacillus fermentation. The downstream recovery process intermediates were analysed using reversed-phase ultra-high-pressure liquid chromatography with electrospray ionisation and high-resolution time-of-flight mass spectrometry detection. To evaluate the pre-treatment agents the following multicriteria was applied i) practical considerations, ii) total amino acid in the precipitated pellet, iii) putative identification of the molecules removed or created by the different treatments, iv) coherence between high quality extracted ion chromatograms (repeatability of DW-CODA) and v) replicate consistency from principal component analysis score values obtained by using the CHEMometric analysis of sections of Selected Ion Chromatograms (CHEMSIC) method. This study presents a generic workflow to find the best pre-treatment for removing bio-macromolecules from biofluids with a multicriteria approach. In our case, the best protein removal strategy for downstream recovery intermediates was acetonitrile precipitation. This method showed high precision, created few artefact peaks compared to simple sample dilution, and mainly removed small peptides.

Role of electrostatic repulsion on colloidal stability of Bacillus halmapalus alpha-amylase.

The colloidal stability of charged particles in suspension is often controlled by electrostatic repulsion, which can be rationalized in a semi-quantitative way by the DLVO theory. In the current study, we investigate this approach towards understanding irreversible protein aggregation, using Bacillus halmapalus alpha-amylase (BHA) as a model protein. Repulsive forces between partly unfolded monomers were shown to strongly affect aggregation. Adding salt, increasing valence of counter ions or decreasing pH in the direction of pI resulted in a shift in the rate-limiting step from association to unfolding as evidenced by a change in aggregation kinetics from second to first-order in protein concentration. Charge screening effects by salts resulted in increased average size of protein aggregates but only moderately affected the secondary structure of protein within the aggregates. Salt and pH effects could be explained within the DLVO framework, indicating that partially unfolded BHA monomers can be modelled realistically as colloids with a random charge distribution.

A differential vapor-pressure equipment for investigations of biopolymer interactions.

The design and performance of an equipment for the measurement of vapor pressures over liquid or solid samples is presented. The equilibrium pressure difference, DeltaP, between a sample and a reference of known vapor pressure is recorded as a function of composition and/or temperature. Through the use of high-accuracy capacitance manometers and a leak-tight system of stainless steel pipes, below-sealed valves and metal-gasket fittings, DeltaP can be measured with a resolution of about 0.5 micro bar (0.05 Pa) in some applications. This sensitivity level, along with other features of the equipment, particularly a "gas-phase titration" routine for changing the cell composition, makes it effective for the investigations of several types of biopolymer interactions. These include isothermal studies of net affinities such as the adsorption of water to proteins or membranes, the preferential interaction of biopolymers with the components of a mixed solvent, the partitioning of solutes between a membrane and the aqueous bulk and the weak, specific binding of ligands to macromolecules. Furthermore, a temperature-scanning mode allows real-time elucidation of such interactions at thermally induced conformational changes in biopolymers. Selected examples of these applications are presented and discussed.