Protein analysis by two-dimensional LC using size-exclusion and reverse phase chromatography with column switching

Over the years, two-dimensional Liquid Chromatography (2D-LC) has played a key role in the field of proteomics for the analysis and isolation of proteins from complex matrices, before their characterization by Mass Spectrometry1,2. In 2D-LC method development, a large number of experimental variables have to be extensively studied to take full advantage of the claimed features of the chromatographic separations3. In the last decade, significant improvements in LC column technology and instrumentation have been made and the introduction of superficially porous particles opened new possibilities in terms of speed and resolution. Here an automated method combining size exclusion with reverse phase chromatography and UV detection is proposed for protein separations in food samples. For the first dimension separation, a size-exclusion column, packed with 3 μm ultra-pure silica particles densely bonded with a proprietary hydrophilic surface chemistry, was used, ensuring high efficiency and resolution. A reversed phase widepore column was used for the second dimension separation, based on core-shell particle technology that provides striking increases in peak capacity and resolution at lower backpressures. Different collection trap systems were placed on a 10-port switching valve and tested to focus the proteins coming from the first dimension column, before their selective elution in the second dimension column. A protein standard mixture of bovine serum albumin, -lactoglobulin and glucose oxidase was used to optimize the gradient chromatographic separation conditions, after evaluating their retention behavior in each single column. Finally, the optimized 2D-LC method was applied to the protein analysis in eggs, infant milk powder and soy flour samples.