Development of a univariate membrane-based mid-infrared method for protein quantitation and total lipid content analysis of biological samples.

Biological samples present a range of complexities from homogeneous purified protein to multicomponent mixtures. Accurate qualification of such samples is paramount to downstream applications. We describe the development of an MIR spectroscopy-based analytical method offering simultaneous protein quantitation (0.25-5 mg/mL) and analysis of total lipid or detergent species, as well as the identification of other biomolecules present in biological samples. The method utilizes a hydrophilic PTFE membrane engineered for presentation of aqueous samples in a dried format compatible with fast infrared analysis. Unlike classical quantification techniques, the reported method is amino acid sequence independent and thus applicable to complex samples of unknown composition. By comparison to existing platforms, this MIR-based method enables direct quantification using minimal sample volume (2 µL); it is well-suited where repeat access and limited sample size are critical parameters. Further, accurate results can be derived without specialized training or knowledge of IR spectroscopy. Overall, the simplified application and analysis system provides a more cost-effective alternative to high-throughput IR systems for research laboratories with minimal throughput demands. In summary, the MIR-based system provides a viable alternative to current protein quantitation methods; it also uniquely offers simultaneous qualification of other components, notably lipids and detergents.

Simultaneous reduction and alkylation of protein disulfides in a centrifugal ultrafiltration device prior to two-dimensional gel electrophoresis.

Reduction and alkylation of protein disulfides prior to IEF, when performed directly in a centrifugal ultrafiltration device, provides an effective means of terminating the alkylation reaction, concentrating the proteins for analysis, and removing ionic impurities that interfere with IEF. When cells were lysed in "buffers" that support the activity of enzymes such as lysozyme and benzonase, the conductivity of the resulting lysate was an order of magnitude higher than when lysis was induced by chaotropic urea detergent solutions. Following reduction and alkylation, the conductivity of both lysates was lowered by ultrafiltration to the 0.1-0.2 mS/cm range in preparation for IEF. The detergent 3-(4-heptyl)phenyl 3-hydroxypropyl dimethylammonio propanesulfonate (C7BzO), which favors the solubilization of proteins, but which interferes with SDS equilibration and second dimension PAGE, was effectively removed by ultrafiltration and exchanged with CHAPS without measurable loss of protein. Disparate protein patterns of Rhodopseudomonas palustris lysates were revealed by two-dimensional gel electrophoresis depending on which reagent was used to induce cell lysis.

Ultrafiltration for proteomic sample preparation.

Proteome analysis represents significant challenges to the existing sample preparation techniques. Traditional methods, such as two-dimensional electrophoresis, typically separate high-molecular-weight proteins while discarding low-molecular-weight species. This approach is well justified considering the complexity of any proteome. However, it is desirable to extract the maximum amount of information from each sample to investigate the entire range of biomolecules. We have demonstrated that ultrafiltration not only improves two-dimensional electrophoresis (2-DE) resolution of the protein fraction but also yields the low-molecular-weight fraction amenable for further analysis by high-resolution mass spectrometry. This approach was successfully adapted to the variety of biological samples including cell and tissue lysates and serum. Therefore, ultrafiltration offers an alternative sample preparation technique that enables more thorough analysis of a proteome.