Recent Advances in Chiral Analysis of Proteins and Peptides

Like many biological compounds, proteins are found primarily in their homochiral form. However, homochirality is not guaranteed throughout life. Determining their chiral proteinogenic sequence is a complex analytical challenge. This is because certain D-amino acids contained in proteins play a role in human health and disease. This is the case, for example, with D-Asp in elastin, β-amyloid and α-crystallin which, respectively, have an action on arteriosclerosis, Alzheimer's disease and cataracts. Sequence-dependent and sequence-independent are the two strategies for detecting the presence and position of D-amino acids in proteins. These methods rely on enzymatic digestion by a site-specific enzyme and acid hydrolysis in a deuterium or tritium environment to limit the natural racemization of amino acids. In this review, chromatographic and electrophoretic techniques, such as LC, SFC, GC and CE, will be recently developed (2018–2020) for the enantioseparation of amino acids and peptides. For future work, the discovery and development of new chiral stationary phases and derivatization reagents could increase the resolution of chiral separations.

Continuous Separation of Model Proteins by Free-Flow Field Step Electrophoresis and its Electrokinetic Basis

Continuous separation of cells, cell membranes, and proteins by electrophoretic techniques such as free-flow zone electrophoresis (FFZE) has a rich history since its introduction sixty years ago by Hannig. However, the results of FFZE, capillary zone electrophoresis, and other similar electrophoretic techniques are limited to analytical scale separations that are not readily extendable to the preparative scale. Moreover, a five- to ten-fold dilution of samples by buffer is common in separations by these techniques. Free-flow field step electrophoresis (FFFSE) is an electrophoretic technique that appears to be suitable for continuous simultaneous preparative separation and concentration and therefore has the power to overcome the above limitations. Here we apply FFFSE to a simple system of model proteins to show proof-of-concept of the technique. A continuous, preparative-scale separation of myoglobin from BSA with a throughput of 20 mg/h and a yield of >98% is shown to be successfully obtained using single-step FFFSE. Most important, it is shown that the preparative FFFSE experiment can be rationally designed, and the results predicted theoretically by use of electrokinetic data recorded in a simple analytical-scale FFZE experiment. This is the first paper to present a theory of separation in FFFSE. The separation is continuous , single-step , and environmental-friendly as no adjuvants are used, with no need for regeneration of components. The electrokinetic basis of the separation appears to be of a general nature. In further research we are testing the limits of the technique, by exploring its extension to more complex systems, and to higher preparative-scale throughputs.

Partial characterization of digestive proteases in Pacific red snapper Lutjanus peru Nichols & Murphy, 1922 (Perciformes: Lutjanidae)

Pacific red snapper (Lutjanus peru) is an important commercial species in Mexico with great aquaculture potential; however, digestive physiology is still unknown. Therefore, the objective of the present work was to characterize the digestive proteases of L. peru juvenile using biochemical and electrophoretic techniques. Results showed a higher acid protease activity than the alkaline proteases, trypsin, chymotrypsin, and leucine aminopeptidase (LAP). The optimum temperature for acid proteases was between 30 to 40°C. Trypsin activity showed two maximum peaks of temperature (30 and 50°C), while alkaline proteases, chymotrypsin, and LAP had optimum temperatures of 50, 50 to 60, and 40°C, respectively. Moreover, the optimum pH of acid proteases was between 2 and 3. Also, alkaline proteases, trypsin, chymotrypsin showed pH optimums at pH 6, 9, and 5, respectively, although LAP showed two optimum pH values at 6 and 9. Acid protease zymogram showed three isoforms, totally inhibited by pepstatin A. Alkaline protease zymogram revealed six bands (125.4, 67.2, 57.9, 48.6, 29.8, and 26.9 kDa), which were inhibited by specific serine-proteases and metalloproteases inhibitors. In conclusion, the main digestion in L. peru depends on stomach proteases, which are characteristic of carnivorous fish, followed by intestinal digestion supported mainly by chymotrypsin.

Skimmed Goat’s Milk Powder Enriched with Grape Pomace Seed Extract: Phenolics and Protein Characterization and Antioxidant Properties

The aim of this research was phenolics and protein characterization and antioxidant properties evaluation of skimmed thermally treated goat’s milk powder enriched with different concentration of grape pomace seed extract (SE). The dominant phenolics in SE were phenolic acids, flavan–3-ols and procyanidins. Different electrophoretic techniques together with UHPLC-MS/MS analysis revealed the presence of phenolics-protein interactions in the samples, mainly procyanidins with whey protein/caseins complexes. Addition of SE into thermally treated goat’s milk significantly improved antioxidant properties of goat’s milk such as TAC, FRP, DPPH• and ABTS•+ scavenging activity. Gallic acid, catechin, and procyanidins mostly contributed to these activities. The schematic representation of phenolics–casein micelles interactions in thermally treated goat’s milk enriched with SE was given. The addition of SE into thermally treated goat’s milk can be a promising strategy in food waste recovery and to enhance the beneficial health effects of goat’s milk-based functional foods.