Centrifugal Filtration Devices to Separate Peptides/Proteins as a Characterization Tool: Good Specificity but Poor Quantification

Centrifugal filtration devices are increasingly being used to separate (bio)molecules thanks to their molar masses. This is partly due to their ease of use, with sample preparation requiring no complicated protocol and often being able to be run as is. Nevertheless, several limitations have been highlighted in the literature, such as proteins’ selectivity according to their molar masses, clogging problems and some post-separation quantification issues. We wanted to test the real potential of the centrifugal filtration devices as a characterization tool, by verifying their specificity using a library of peptides and proteins. The impact of the peptide charge on separation performance as well as the impact of biological material concentration and centrifugation rate were evaluated using three membranes with different cut-offs. A good specificity was confirmed: biological materials that have molar masses which are below the membrane cut-off can pass almost completely through the membrane, while those with molar masses above the membrane cut-off are almost fully retained. The findings proved more problematic when protein mixtures were analyzed: despite still having good specificity, biological materials with molar masses below the membrane cut-off only partially crossed the membranes, due to clogging effects, making quantification of the initial mixture incorrect. It is therefore essential to be very careful when working with centrifugal filtration devices, as their use requires precise and relevant controls, without necessarily being able to optimize the separation of mixtures. Thus, this remains far from an easy and ready-to-use system.