An automated food protein isolation approach on preparative scale by two‐dimensional liquid chromatography with active modulation interface

Abstract

In this work, an automated 2D-LC approach for protein isolation from egg samples on preparative scale is proposed. The method is based on the use of a C18 guard column installed in a switching valve to focus the proteins coming from the first dimension column, before their elution in the second column. For the first dimension separation, a size-exclusion column, packed with 3 m ultrapure silica particles was used. An RP column based on core-shell technology was used for the second dimension separation. A standard mixture of BSA, ␤-lactoglobulin, and glucose oxidase, chosen as a protein model system, was used to optimize the chromatographic separation conditions. The fully automated workflow allowed to isolate, in a single-chromatographic analysis, a protein amount of 50 g for each peak fraction, with a total time of 15 min for the first separation and additional 30 min of the second separation for each trapped protein. The final aim was the development of proper analytical tools for protein isolation from foodstuffs to be used for the molecular identification by MS, as well as for biotherapeutic uses, allergy testing, and large-scale investigations in biological systems.

Recent stationary phase‐based fractionation strategies in proteomic analysis

“…Then, after a 2-minute wash of the trapping column with water + 0.1% TFA mobile phase, the flow was delivered in a back-flush to release the trapped proteins to the analytical column where they were separated by gradient elution RPLC. With the aid of an Automatic Fraction Collector at the end of the RP column, these two reported systems consisted of automated methods to purify proteins on food samples based on the SEC-RP separation to be further characterized by MS [50,51].…”

“…Although not used for routine bio-purification studies like offline approaches, the online approaches are more interested in the proof of concept of new analytical instrumentation. Nardielloťs group employed UV detection to monitor the orthogonal SEC-RP separation of proteins from eggs (in the first work) and soy flour (in the second work) [50,51]. The online setup consisted of multiport switching valves connected with two analytical pumps, and one RP trapping column between 1 D and 2 D to reduce dead volumes and band-broadening effects.…”

Comprehensive and heart‐cutting multidimensional liquid chromatography–mass spectrometry and its applications in food analysis

“…From the analytical point of view, the characterization of biomolecules is quite challenging due to their great heterogeneity, thus, several 2DLC–MS methods have been described for the characterization of proteins in food, being the combination of SEC and RP the most frequently used mechanism due to its high orthogonality. Therefore, SEC mode is usually employed in the 1 D to separate proteins according to their size (whose elution order is directly related to their molecular weight), while RP mode allows the separation of proteins on the basis of hydrophobic chain length, being this mechanism used in the 2 D due to its compatibility with MS. A SEC‐RPLC method was developed by Melfi et al (2019), employing a stop‐and‐go approach for the isolation of intact proteins in egg samples by UV‐IT‐MS detection. An active modulation was proposed by using a RP guard column installed in a switching valve as a trapping interface.…”

Less Configuration and More Dimensionality: Preparative Heart-Cut Multidimensional Liquid Chromatography Based on Trapping Arrays

“…Zhu , has developed a trapping array-based preparative 2D-LC system for the isolating of herbal medicines. And few preparative 2D-LC configurations run in heart-cut mode, with one − or two fractions ,− were transferred to 2 D separations. Although preparative 2D-LC methods have been established and are quite powerful, exhaustive separations of very complex samples are still challenging. , This has motivated researchers to investigate the potential benefits of adding more LC columns to a 2D-LC system .…”