Enhancing online protein isolation as intact species from soy floursamples by actively modulated two-dimensional liquidchromatography (2D-LC)

tIn this study, an enhanced fully automated approach is described for the protein isolation from soyflour samples by two-dimensional liquid chromatography with active modulation interface. The useof two multi-port switching valves is proposed to on-line connect the first to the second dimensioncolumn, thus overcoming the problems associated with the re-mixing effects and incompatibility ofeluent composition and pH. A 5-cm long C4 analytical column installed in the interface device allows tofocus the proteins coming from the first column (size exclusion chromatography), before their selectiveelution in the second column (reversed-phase). A trap washing step was included in the total workflow,as a desalting step to remove buffer residues from the eluent of the first column and to enhance thechromatographic performances of the second column. The experimental conditions were optimized byanalyses of mixed standard solutions of bovine serum albumin, glucose oxidase, immunoglobulin A,thyroglobulin and myoglobin. Then, the optimized 2D-LC method was applied to the protein analysis inextracts of soy flour, known worldwide as one of the major food allergen sources, with the final aim torecovery sufficient protein amounts for the molecular characterization and the assessment of the patternof allergenic components.