Analytical strategies for the characterization, identification and quantification of peptides and proteins of interest in the prevention and understanding of hypertension (2023)

Hypertension is a serious health problem and is considered one of the causes of cardiovascular and kidney diseases. Despite the prevalence of hypertension, half of the people affected are unaware that they have it. In addition, studies carried out on the most important causes of death in the world predict an increase in the contribution of cardiovascular diseases. As the symptoms of hypertension can go unnoticed and rarely manifest themselves, the prevention and treatment of hypertension are considered of great importance in modern society. The treatment of hypertension decreased the incidence of cardiovascular accidents. However, although the use of synthetic drugs has been decisive in this decline, these drugs often have side effects.

An interesting alternative to these drugs are some peptides found naturally in certain foods. In fact, a basic strategy to improve cardiovascular health is to modify diet and lifestyle, since diet is one of the factors that most influence human health. Among the bioactive compounds found in foods, bioactive peptides have received great attention in recent years. In particular, antihypertensive and antioxidant peptides are the most studied and have demonstrated their positive effect on cardiovascular health. Indeed, antihypertensive peptides can inhibit ACE activity and lower blood pressure. In turn, antioxidant peptides prevent oxidative stress that can initiate and promote the onset of hypertension. Bioactive peptides can be natural ingredients in foods or originate from food proteins of which they are a part by in vivo or in vitro procedures. Gastrointestinal digestion itself constitutes an in vivo procedure while the in vitro procedure involves the hydrolysis of proteins by the action of enzymes or microorganisms added to the food. In the case of processed products, bioactive peptides are released from proteins during food processing (cheese, yogurt, kefir, etc.).

To date, research related to antihypertensive peptides and antioxidants has focused primarily on animal foods such as milk, dairy products and meat. However, bioactive peptides of plant origin, although less studied, often have superior activities. Corn and soy are examples of sources of highly active bioactive peptides. Soy infant formulas (SBIFs) are a very interesting alternative to milk and derivatives for children with intolerance or allergy to some constituents of milk, with feeding problems or belonging to vegetarian families. However, compared to milk and dairy products, SBIFs have received little research on bioactive peptide content. These infant formulas are made from soy protein isolates that contain about 90% protein. During their preparation, they are subjected to intense heat or protein hydrolysis. Thus, SBIFs may naturally contain potentially bioactive peptides with beneficial health effects in addition to their nutritional benefits. In this work, SBIFs were chosen as a potential source of bioactive peptides. Four different methods have been proposed to extract peptides from SBIFs.

In this work, an analytical methodology was developed for the simultaneous determination of LRP, LSP and LQP peptides from [alpha]-zeins present in corn kernels. Additionally, an analytical methodology was developed by HPLC-Q-ToF-MS for the determination of the three mentioned peptides in maize. To carry out the determination of the VLIVP peptide in soy, this work also developed an analytical methodology using capillary HPLC coupled to an ion trap mass spectrometry system (capillary HPLC-IT-MS).

Finally, in this work, a SRM method (selected reaction monitoring) using triple quadrupole mass spectrometry (QqQ) detection was developed to evaluate the content of protein kinase isoforms PKA, PKG and CaMKII in different tissues of rats. In summary, this work investigated for the first time the presence of native bioactive peptides present in SBIFs.

These studies allowed obtaining a broad view of the potential of the bioactive peptides present in these baby foods and, at the same time, observing great differences between them, contributing to increase knowledge of the real nutritional value of these foods, as well as their physiological and biological effects. On the other hand, analytical methodologies were also developed for the determination of highly potent ACE inhibitor peptides in corn and soybean crops. These methods were characterized and applied in the analysis of different varieties of these crops. The results obtained have a significant potential in the field of food science closely related to the area of ​​biomedicine.

Finally, another aspect investigated in this work was the determination of protein isoforms of high cardiovascular interest in different tissues of rats. Although an appropriate SRM method has been developed, the complexity of the sample did not allow reliable quantification of the PKA, PKG and CaMKII isoforms, therefore further studies are needed to overcome this difficulty. Once overcome, this strategy could have a great impact on the investigation of the molecular mechanisms involved in the cardiovascular system.

Hypertension is recognized as a serious health problem worldwide, being considered one of the main causes of several cardiovascular and kidney diseases. Despite the prevalence of hypertension, about half of those affected are unaware of their condition. In addition, forecasts on the main causes of death in the world anticipate an increasing participation of cardiovascular diseases. Coupled with the fact that the symptoms of hypertension are hidden and rarely occur, the prevention, treatment and mitigation of hypertension are given high priority in modern society.

General knowledge about the cardiovascular system and hypertension is fairly well established. However, some information related to some molecular mechanisms of the cardiovascular system is missing. It is well known that among the various systems that control the blood pressure level, the renin-angiotensin system plays the central role. In fact, angiotensin I converting enzyme (ACE), one of the main players 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 an important role in controlling the blood pressure level. Consequently, synthetic antihypertensive drugs with the ability to inhibit ACE activity have been mainly used. Other important regulatory proteins in the cardiovascular system are PKA, PKG and CaMKII. These proteins have different isoforms and several scientific works have suggested great differences between them. However, its exact expression levels in body organs are still unknown, limiting knowledge about its important role in the cardiovascular system.

Treatment of hypertension has been shown to decrease the occurrence of several cardiovascular events. However, although the use of synthetic drugs has been decisive for this decrease, they usually produce side effects. An interesting alternative to synthetic drugs are the peptides found naturally in some foods. In fact, the basic strategy to improve cardiovascular health is to change diet and lifestyle, as nutrition is one of the main factors that influence human health. Among the bioactive compounds in foods, bioactive peptides are attracting much attention. Specifically, antihypertensive and antioxidant peptides are the most commonly reported and have been shown to contribute positively to cardiovascular health. In fact, while antihypertensive peptides can inhibit ACE activity and effectively lower blood pressure levels, antioxidant peptides prevent oxidative stress that can initiate and promote the development of hypertension. Bioactive peptides can be natural food ingredients or they can be released from the original food proteins using in vivo or in vitro approaches. The in vivo strategy is gastrointestinal digestion itself, while the in vitro approach involves protein hydrolysis by non-specific enzymes or microorganisms added to food. Special attention deserves elaborate products. In this case, bioactive peptides are released from proteins during food processing (eg cheese, yogurt, kefir). To date, research on antihypertensive peptides and antioxidants has mainly been devoted to foods of animal origin, such as milk, dairy products and meat. However, less studied plant-derived bioactive peptides have often shown more potent activities. Corn and soy are exceptional examples of sources of very potent bioactive peptides.

The presence of soy-based infant formulas (SBIF) on the market is significant, as they constitute an alternative to dairy products and milk for infants with intolerance or allergy to some components of milk, with feeding problems or from vegan families. However, compared to milk and dairy products, they have not been widely exploited for their bioactive peptide content. Modern SBIFs are based on soy protein isolate, which contains about 90% protein. During preparation by the manufacturer, SBIFs are subjected to intense heat and/or protein hydrolysis. Therefore, SBIFs may naturally contain potential bioactive peptides that may exert specific health effects in addition to their nutritional benefits.

In this research work, SBIFs were selected as a potential source of bioactive peptides. Four different methods have been proposed to extract peptides from SBIF. Antioxidant peptide capacity was determined using three different antioxidant assays, while antihypertensive capacity was assessed by measuring the ability to inhibit ACE in vitro. Direct ultrafiltration through 10 kDa Mwco filters provided the extract with the highest level of peptide concentration and antioxidant capacity. The SBIF extracts were then fractionated using different Mwco filters and the antioxidant and antihypertensive capacity of these fractions was evaluated. Fractions of 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. OFFGEL isoelectric focusing proposed another fractionation of this fraction. However, the ampholytes needed to establish the pH gradient for separation by isoelectric focusing interfered with the antioxidant assays used in this work. To remove these ampholytes, several strategies have been proposed. The chromatographic separation with a monolithic column allowed the removal of ampholytes from the investigated samples. However, the individual OFFGEL fractions showed much lower antioxidant capacities than the output sample, suggesting a synergistic effect between the antioxidant peptides. Therefore, the OFFGEL separation step has been removed from the survey workflow. On the other hand, the highest ACE inhibitory capacity was observed in fractions of 3-5 kDa and below 3 kDa. In these cases, the subsequent separation of the fractions by isoelectric focusing was not considered. ACE inhibitor peptide fractions were then identified by HPLC coupled to a quadrupole time-of-flight mass spectrometer (Q-ToF-MS) and PEAKS software. Data analysis showed a low selectivity in ultrafiltration fractionation. Then, the fractions with the highest ACE inhibitory capacity (3-5 kDa and below 3 kDa) and antioxidant (5-10 kDa) were submitted to simulated gastrointestinal digestion with pepsin and pancreatin. The results showed that the antioxidant capacity was insignificantly changed after simulated gastrointestinal digestion, while the ACE inhibitory capacity of the peptide fractions decreased. Peptides obtained after the simulated gastrointestinal digestion procedure were also identified. More than 120 peptides were identified in each antioxidant fraction, where 42 peptides were common to all SBIFs. The VAWWM peptide was found in all SBIF antioxidant fractions studied. This peptide is part of the sequence of soystatin (VAWWMY), a soy peptide previously reported as a strong cholesterol absorption inhibitor and bile acid binder. In the case of the ACE inhibitor fractions, there were 13 peptides in the 3-5 kDa fraction and 20 peptides in the sub-3 kDa fraction that could support the gastrointestinal digestion process.

Interestingly, the RPSYT peptide appeared in all infant formulas and showed antioxidant and ACE inhibitory abilities. 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 indicated above, corn and soy are attractive sources of bioactive peptides. Special attention deserves the exceptionally potent ACE inhibitor peptides LRP (IC50 = 0.29 µM), LSP (IC50 = 1.7 µM) and LQP (IC50 = 2.0 µM), obtained by digestion with α-thermolysin. - corn zeins, and the peptide VLIVP (IC50 = 1.69 µM) found in hydrolyzed 11S soybean glycinin protease P. The ACE inhibitory capacity of these peptides is much higher than that of the known peptides VPP (IC50= 9.13 µM) or IPP (IC50= 5.15 µM) from milk. Considering the high dose dependence of ACE inhibitor peptides and the differences in protein content observed between corn cultures, the need for analytical methodologies for the quantitative evaluation of these peptides is evident. However, despite the enormous interest that exists in the determination of ACE inhibitor peptides in foods, the literature on the subject is quite scarce.

In this work, an analytical methodology was developed for the simultaneous determination of LRP, LSP and LQP peptides from α-zeins contained in whole corn kernels. Existing extraction methods for obtaining α-zeins focus on corn protein concentrates such as corn gluten meal rather than corn kernels with a much lower protein concentration. Therefore, a method using high-intensity focused ultrasound for the extraction of α-zein proteins from whole corn kernels was developed. Furthermore, purification of α-zein extracts by acetone precipitation has also been proposed. The recoveries of proteins extracted from different cultures were close to 100%. An adequate buffer was selected that would allow the solubilization of corn proteins and that would be compatible with thermolysin activity. The digestion procedure of α-zein extracts by thermolysin was optimized. The presence of the three antihypertensive peptides in the digested extracts was confirmed by HPLC-Q-ToF-MS analysis and by comparison with peptide standards. Separation conditions were optimized in a new stationary phase of molten core and the antihypertensive capacity of corn cultures was evaluated by HPLC-UV.

In this research work, the determination of the referred peptides in corn grains by HPLC-Q-ToF-MS was also carried out. The stability of standard and sample solutions was studied. Different MS parameters were optimized to avoid spontaneous fragmentation of peptides in the source. The optimization of these parameters not only decreased the spontaneous fragmentation in the ESI source, but also allowed to increase the sensitivity. Two different strategies based on FASP (filter-assisted sample preparation) and SPE (solid phase extraction) have been proposed to remove urea from digested extracts due to its interference in 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 to the quantification of specific peptides. The developed method was characterized by evaluating linearity, limits of detection and quantification, repeatability, intermediate precision and recovery. A study was also carried out on the existence of matrix interferences. The developed method was applied to the quantification of LRP, LSP and LQP peptides in different maize lines using the calibration method of standard additions. The results showed great differences in the contents of the three peptides between the corn lines studied. In general, the most abundant peptide was LSP followed by LQP while the LRP peptide had the lowest content despite being the most antihypertensive.

To evaluate the VLIVP content in soybean cultures, an analytical methodology using capillary HPLC and ion trap mass spectrometry (capillary HPLC-IT-MS) was developed. First, a previously developed method for extracting protein from soybean crops was implemented. However, the selected extraction method was time consuming and involved a higher precipitation of 11S glycinine at its isoelectric point. The application of high-intensity focused ultrasound and the optimization of the conditions made it possible to reduce the extraction time from 2 h to 2 min. It was found that the further isoelectric precipitation of the 11S glycinin fraction was not quantitative and therefore this step was rejected. The VLIVP peptide was identified in protease P hydrolyzate of soy whole proteins by capillary HPLC-IT-MS in MS and MS/MS modes. Injection of the peptide template using MS/MS showed that the transition 540.4 425.3 was the dominant one. Several chromatographic conditions (elution gradient, ion pairing reagent and separation temperature) were optimized, enabling the separation of the VLIVP peptide in just 7 min. In addition, the soy protein protease P hydrolyzate was diluted to eliminate ionization interference from the sample. Digestion with protease P enzyme has been optimized to obtain better digestion yield and reduce digestion time. To improve sensitivity, several 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, with the highest peptide content in the Polish soybean variety.

Finally, a triple quadrupole (QqQ) SRM (selected reaction monitoring) assay was developed to estimate the content of PKA, PKG, and CaMKII kinase isoforms in different tissues of mice. In silico digestion simulation, previous results and database searches (PeptideAtlas and BLAST) allowed the preliminary selection of an appropriate set of proteotypic peptides. A new strategy was employed using an Orbitrap-Velos MS with an HCD (Higher Energy Collision Dissociation) fragmentation system. Digested tissues containing enriched target protein kinases were analyzed. The results allowed confirming a large number of theoretically selected proteotypic peptides and defining the most likely list of transitions. Said set of peptides/transitions was verified in QqQ, allowing the creation of a final SRM assay. Analysis of highly labeled peptides in digested kidney tissues allowed the validation of a selected set of peptides and transitions. Sensitivity was increased by programming the method into the HPLC run and optimizing the collision energy for each peptide. The SRM assay was applied to heart, liver, and kidney digested lysates that exhibited a dynamic range that was not high enough to determine all target protein isoforms. Previous separation of the lysate by sodium dodecyl sulfate and polyacrylamide gel electrophoresis (SDS-PAGE) reduced the complexity of the cardiac lysate. However, such an approach showed a lack of reproducibility when applied to a large set of cardiac lysates. Additional studies were suggested to reduce sample complexity.

In summary, this work evaluated for the first time the presence of native bioactive peptides in SBIF. Native peptides present in SBIFs were studied for the first time. These studies showed a broad view of potential bioactive peptides in SBIFs. These results improved knowledge about the real nutritional value and the physiological and biological effects of SBIF. On the other hand, methods were developed for the determination of high potency ACE inhibitor peptides in corn and soybean crops. These methods were successfully characterized and applied to the analysis of different crop varieties. The results have great potential importance in food research strictly related to the biomedical area. Finally, the quantification of protein isoforms of high cardiovascular interest in different tissue samples was also investigated. Although a suitable SRM assay was developed, the complexity of the sample did not allow reliable quantification of specific PKA, PKG and CaMKII isoforms. New studies need to be carried out to overcome this problem and, when successful, this approach can have a great impact on the general knowledge about the molecular mechanisms of the cardiovascular system.