Analytical strategies for the characterization, identification, and quantification of peptides and proteins of interest in the prevention and understanding of hypertension

Résumé

Hypertension is recognized as a serious worldwide health problem and it is considered as a leading cause of various cardiovascular and kidney diseases. Despite the prevalence of hypertension, around half of affected individuals are unaware of their condition. Additionally, forecasts concerning major causes of worldwide deaths predict the growing participation of cardiovascular disease. Together with the fact that symptoms of hypertension are hidden and rarely occur, the prevention, treatment, and mitigation of hypertension receive a high priority in modern society. General knowledge on cardiovascular system and hypertension is quite well established. However, some pieces of information related to some molecular mechanisms in cardiovascular system are absent. It is well known that among various systems controlling blood pressure level, the renin-angiotensin system plays the pivotal role. Indeed, angiotensin I converting enzyme (ACE), main player in the renin-angiotensin system, converts angiotensin I into the vasoconstrictor angiotensin II and, at the same time, deactivates the vasodilator bradykinin. Therefore, this enzyme plays a significant role in the control of blood pressure level. Consequently, synthetic antihypertensive drugs with capacity to inhibit ACE activity have been mostly used. Other important regulatory proteins in cardiovascular system are PKA, PKG, and CaMKII. These proteins present different isoforms and various scientific reports have suggested great differences among them. Nevertheless, their exact expression levels in body organs is still unknown, which limits the knowledge on their important function in the cardiovascular system. The treatment of hypertension has shown to decrease the occurrence of various cardiovascular events. Nevertheless, although the use of synthetic drugs has been decisive for this decrease, they usually cause side effects. An interesting alternative to synthetic drugs are peptides found naturally in some foods. In fact, the basic strategy to improve cardiovascular health is to change diet and lifestyle since nutrition is one of the main factors influencing human health. Among food bioactive compounds, bioactive peptides are attracting great attention. Specifically, antihypertensive and antioxidant peptides are the most commonly reported and have proved to positively contribute to cardiovascular health. Indeed, while antihypertensive peptides can inhibit ACE activity and effectively decrease blood pressure level, antioxidant peptides prevent oxidative stress that can initiate and promote the development of hypertension. Bioactive peptides can be natural ingredients of foods or can be released from parent food proteins by in vivo or in vitro approaches. The in vivo strategy is the gastrointestinal digestion itself while the in vitro approach involves protein hydrolysis by non-specific enzymes or microorganisms added to foods. Special attention deserves processed products. In this case, bioactive peptides are released from proteins during food processing (e.g. cheese, yoghurt, kefir). Until date, the investigation concerning antihypertensive and antioxidant peptides was mainly devoted to animal origin foods like milk, dairy products, and meat. However, less studied vegetable origin bioactive peptides have frequently shown more potent activities. Maize and soybean are exceptional examples of sources of highly potent bioactive peptides. The presence of soybean based infant formulas (SBIFs) in the market is significant since they constitute an alternative to dairy products and milk for infants with intolerance or allergy to some milk constituents, with feeding problems or coming from vegan families. Nevertheless, in comparison with milk and dairy products, they have not been much explored for their content in bioactive peptides. Modern SBIFs are based on soybean protein isolate that contains around 90% of proteins. During manufacturer preparation, SBIFs are subjected to intense heat and/or protein hydrolysis. Thus, SBIFs can naturally contain potential bioactive peptides that might exert specific health effects apart from their nutritional benefits. In this research work, SBIFs were selected as a potential source of bioactive peptides. Four different methods were proposed to extract peptides from SBIFs. The antioxidant peptide capacity was determined using three different antioxidant assays while antihypertensive capacity was evaluated by measuring the capacity to inhibit ACE in vitro. The direct ultrafiltration through 10 kDa Mwco filters provided the extract with the highest peptide concentration level and antioxidant capacity. SBIF extracts were next fractionated using different Mwco filters and the antioxidant and antihypertensive capacity of these fractions were evaluated. Fractions from 5-10 kDa, 3-5 kDa, and below 3 kDa were obtained and studied. The highest antioxidant capacity, in most cases, was detected in the 5-10 kDa peptide fractions. A further fractionation of this fraction was proposed by OFFGEL isoelectrofocusing. Nevertheless, ampholytes necessary to establish pH gradient for the isoelectrofocusing separation interfered with the antioxidant assays employed in this work. In order to remove these ampholytes various strategies were proposed. Chromatographic separation with a monolithic column enabled to remove the ampholytes from the investigated samples. However, individual OFFGEL fractions showed much lower antioxidant capacities than output sample, suggesting a synergistic effect among antioxidant peptides. Therefore, the OFFGEL separation step was removed from the investigation workflow. On the other hand, the highest ACE inhibitory capacity was observed in fractions from 3-5 kDa and below 3 kDa. In these cases, no additional separation of fractions by isoelectrofocusing was considered. ACE inhibitory peptide fractions were next identified by HPLC coupled to quadrupole time of flight mass spectrometer (Q-ToF-MS) and PEAKS software. The analysis of data showed a poor selectivity in the ultrafiltration fractionation. Then, fractions presenting the highest ACE inhibitory (3-5 kDa and below 3 kDa) and antioxidant (5-10 kDa) capacities were submitted to a simulated gastrointestinal digestion with pepsin and pancreatin. Results showed that the antioxidant capacity changed negligibly after the simulated gastrointestinal digestion while the ACE inhibitory capacity of peptide fractions decreased. Peptides obtained after the simulated gastrointestinal digestion procedure were also identified. More than 120 peptides were identified in every antioxidant fraction where 42 peptides were common for all SBIFs. The peptide VAWWM was found in all the studied SBIF antioxidant fractions. This peptide is a part of the sequence of soystatin (VAWWMY), a soybean peptide previously reported as a strong cholesterol absorption inhibitor and bile acid binder. In the case of the ACE inhibitory fractions, there were 13 peptides in the fraction from 3-5 kDa and 20 peptides in the fraction below 3 kDa that could stand the gastrointestinal digestion process. Interestingly, RPSYT peptide appeared in all infant formulas and showed both antioxidant and ACE inhibitory capacities. Therefore, this peptide was synthesized and further characterized. These studies revealed its resistance to gastrointestinal enzymes and high processing temperatures, its moderate antihypertensive activity, and its potent antioxidant activity. As previously stated, maize and soybean are attractive sources of bioactive peptides. A special attention deserves exceptionally potent ACE inhibitory peptides LRP (IC50 = 0.29 µM), LSP (IC50 = 1.7 µM), and LQP (IC50 = 2.0 µM), obtained by the thermolysin digestion of maize α-zeins, and peptide VLIVP (IC50 = 1.69 µM) found in the protease P hydrolysate of 11S soybean glycinin. ACE inhibitory capacity of these peptides is much higher than that of known peptides VPP (IC50= 9.13 µM) or IPP (IC50= 5.15 µM) from milk. Taking into account the highly dosage dependence of ACE inhibitory peptides and the differences in protein content observed among maize crops, it is clear the need for analytical methodologies for the quantitative assessment of these peptides. Nevertheless, despite the huge interest existing to determine ACE inhibitory peptides in foods, the literature concerning this area is quite scarce. In this work, an analytical methodology for the simultaneous determination of LRP, LSP, and LQP peptides from α-zeins contained in whole maize kernels was developed. Existing extraction methods to obtain α-zeins were focused on maize protein concentrates such as corn gluten meal and not on maize kernels with much lower protein concentration. Therefore, a method using high intensity focused ultrasounds for the extraction of α-zein proteins from whole maize kernels was developed. Furthermore, the purification of α-zein extracts by acetone precipitation was also proposed. The recoveries of proteins extracted from different crops were close to 100%. A suitable buffer enabling the solubilization of maize proteins and that was compatible with thermolysin activity was selected. The digestion procedure of α-zein extracts by thermolysin was optimized. The presence of the three antihypertensive peptides in the digested extracts was confirmed using HPLC-Q-ToF-MS analysis and by comparison with peptide standards. Separation conditions in a novel fused-core stationary phase were optimized and the antihypertensive capacity of maize crops was evaluated by HPLC-UV. The determination of the above-mentioned peptides in maize kernels by HPLC-Q-ToF-MS was also carried out in this research work. The stability of the standard and sample solutions was studied. Different MS parameters were optimized to avoid spontaneous in source fragmentation of peptides. The optimization of these parameters not only decreased the spontaneous fragmentation in the ESI source but also enabled to increase sensitivity. Two different strategies based on FASP (filter aided sample preparation) and SPE (solid phase extraction) were proposed to remove urea from digested extracts due to its interference with MS detection. Appropriate EIC (extracted ion chromatogram) signals (at 193.1315 m/z and 385.2558 m/z for LRP, at 316.1867 m/z for LSP, and at 357.2132 m/z for LQP) were monitored for the quantification of targeted peptides. The developed method was characterized by evaluating linearity, limits of detection and quantitation, repeatability, intermediate precision, and recovery. A study on the existence of matrix interferences was also performed. The developed method was applied to the quantification of LRP, LSP, and LQP peptides in different maize lines using the standard additions calibration method. Results demonstrated great differences in the three peptides contents among the studied maize lines. In general, most abundant peptide was LSP followed by LQP while LRP peptide showed the lowest content despite being the most antihypertensive. In order to evaluate VLIVP content in soybean crops, an analytical methodology using capillary HPLC and ion trap mass spectrometry (capillary HPLC- IT-MS) was developed. A previously developed method was firstly implemented to extract proteins from soybean crops. Nevertheless, the selected extraction method was time consuming and involved a further precipitation of 11S glycinin at its isoelectric point. Application of high intensity focused ultrasounds and the optimization of conditions permitted to decrease the extraction time from 2 h to 2 min. Further isoelectric precipitation of the 11S glycinin fraction resulted non quantitative and, thus, this step was rejected. Peptide VLIVP was identified in the protease P hydrolysate of whole soybean proteins using capillary HPLC-IT-MS in MS and MS/MS modes. The injection of the peptide standard using MS/MS showed that the transition 540.4 425.3 was the dominant. Various chromatographic conditions (elution gradient, ion-pairing reagent, and separation temperature) were optimized being possible the separation of VLIVP peptide within just 7 min. Moreover, the protease P hydrolysate of soybean proteins was diluted to remove sample ionization interferences. The digestion with protease P enzyme was optimized to obtain a better digestion performance and to reduce the digestion time. In order to improve sensitivity, various MS parameters were also optimized. The methodology was characterized by the evaluation of linearity, limits of detection and quantification, matrix interferences, precision, and recovery. The developed method was applied to the analysis of five different soybean crops showing the highest peptide content in the soybean variety from Poland. Finally, an SRM (selected reaction monitoring) assay using triple quadrupole (QqQ) was developed to estimate the content of PKA, PKG, and CaMKII kinase isoforms in different rat tissues. In-silico digestion simulation, previous results, and database searches (PeptideAtlas and BLAST) enabled the preliminary selection of an appropriate set of proteotypic peptides. A new strategy using an Orbitrap-Velos MS with a HCD (higher energy collision dissociation) fragmentation system was employed. Digested tissues containing enriched targeted protein kinases were analyzed. Results allowed to confirm a great number of theoretically selected proteotypic peptides and to define the most probable list of transitions. Such set of peptides/transitions was verified on the QqQ enabling to create a final SRM assay. Analysis of heavily labeled peptides in kidney digested tissues allowed to validate selected set of peptides and transitions. Sensitivity was increased by scheduling the method over the HPLC run and by the optimization of collision energy for every peptide. SRM assay was applied to heart, liver, and kidney digested lysates showing a dynamic range not high enough to determine all targeted proteins isoforms. Previous lysate separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) allowed decreasing heart lysate complexity. Nevertheless, such approach showed a lack of reproducibility when applying to a big set of heart lysates. Additional studies to low down sample complexity were suggested. Summarizing, this work has evaluated for the first time the presence of native bioactive peptides in SBIFs. Native peptides present in SBIFs have been studied for the first time. These studies showed a broad view of potential bioactive peptides in SBIFs. These results have improved the knowledge on real nutritional value and physiological and biological effects of SBIFs. On the other hand, methods to determine highly potent ACE inhibitory peptides in maize and soybean crops were developed. These methods were successfully characterized and applied to the analysis of different crop varieties. The results have a great potential importance in food research strictly connected with the biomedical field. Finally, the quantification of protein isoforms of high cardiovascular interest in various tissue samples was also investigated. Although an appropriate SRM assay was developed, the complexity of sample did not enable a reliable quantification of targeted PKA, PKG, and CaMKII isoforms. Additional studies to overcome this issue must be conducted and, when successful, this approach could have a huge impact on the overall knowledge on molecular mechanisms of the cardiovascular system.