Once defined the protein target, the amino acid sequence is obtained from databases. Then, conserved sequence motifs should be identified if a cross-reactive antibody is desired, or non-conserved regions if a differential antibody is desired. It is also preferable to have a 3D structure of the protein, which can be obtained through homology modeling, in case of not having an experimental structure. This characterization is complemented by the prediction of antigenic sites; this can be done automatically by accessing different servers, such as:

Emboss antigenic [37]: https://www.bioinformatics.nl/cgi-bin/emboss/antigenic

Additionally, several resources regarding T cell epitope prediction servers are described in the review by Soria-Guerra et al. [40]. The epitope determination is complemented by the analysis of 3D structure, choosing the sequences favoring those found in flexible and exposed regions. 3D structures could be obtained from databases, if they have been experimentally determined, or by homology modeling, which can be obtained through multiple servers such as:

I-TASSER [41]: https://zhanggroup.org/I-TASSER/

The chosen sequences are then chemically synthesized, by fluorenylmethyloxycarbonyl (Fmoc) strategy, using tea-bag protocol and are characterized and purified [23, 45]. At this point it is important to note that there are two strategies that guarantee a good antigenic response:

The generation of the synthetic peptide with cysteine residues in the amino and carboxy terminals, allows polymers of the original sequence to be obtained, guaranteeing better recognition of it in the model in which the antibodies are generated (mice or rabbits) [33]. In this regard, the polymerization of the peptides is performed by oxidation at a low concentration (4 mg/mL), to prevent the formation of high molecular weight polymers, which negatively affect the solubility of the peptide.

These two strategies make it possible to avoid the use of carrier proteins.

Synthetic peptides have been used to generate polyclonal antibodies mainly in mice and rabbit models. As mentioned before, polymers of synthetic peptides are used in the formulation. The inoculum of the peptide with FIS is prepared at a 1 mg/mL concentration and the FIS peptide is added at a 1:1 proportion with respect to the peptide. The first dose is administered with complete Freund’s adjuvant, and then two boosts are given before obtaining ascites fluid or blood for antibody purification. Peptide doses are commonly 50–100 μg per mouse and 300–500 μg for rabbits, while the purification of antibodies is conducted through immunoaffinity chromatography, employing cyanogen bromide-activated Sepharose. Finally, the validation is made by dot blot and ELISA using the epitope-peptide, and western blot using protein extract from a tissue that expresses the protein of interest.

After the synthesis and cleavage, the lyophilized peptides were dissolved in water at 1 mM concentration and in 30% trifluoroethanol (TFE), and their corresponding CD spectra were measured at different temperatures using a CD spectrometer. Each spectrum was the average of three scan repetitions in continuous scanning mode with 100 nm/min scanning speed with a response time of 2 seconds.

As described in previous work [50–53, 56], the antimicrobial test was performed using Gram+ and Gram– bacterial strains from ATCC or collection to determine their activity. Minimal bactericidal concentration (MBC) or minimal inhibitory concentration (MIC) were determined by the microplate dilution assay. The different bacterial strains were grown to a logarithmic phase at optimum culture media and temperature. Peptides were probed at less than 100 µM concentration and the use of serial dilutions.

To determine the peptides toxicity two kinds of assays were performed: cytotoxicity and hemolysis [32, 52, 53, 56].

The cytotoxicity of the peptides can be determined by exposing cell monolayers, such as Chinook salmon embryonic cells (CHSE-214) to homopeptide concentrations covering values at least ten times higher than MBC. Cell viability was determined by the trypan blue exclusion technique.

The hemolysis assay was performed with blood samples, from healthy human donors or animals, collected in tubes with anticoagulant. A separation of plasma and cells was made by centrifugation at 2,000 × g for 10 min at 4°C. The supernatant plasma was discarded, and the pellet was washed and resuspended in phosphate buffer saline (PBS) to obtain an approximate concentration of 6 × 10⁸ cells/mL. At this point, the peptides to be tested were added in concentrations at least up to 10 times their MBC. A solution of 0.5% triton X-100 in PBS was used as a positive control, while PBS alone was used as a negative control. For the measurement of hemolysis, the samples are centrifuged at 3,000 × g for 5 min. Supernatant samples were taken and their absorbance was measured in a spectrophotometer at 540 nm. The percentage of hemolysis was calculated according to the following equation:

where: A_s = absorbance of the sample; A_100% = absorbance of the positive control; A_0% = absorbance of the negative control.

The peptides interacting with bacterial strains can be also detected by confocal laser scanning microscopy using fluorophore-labeled peptide, together with DNA fluorophore probes [51, 57, 58]. Cultures of bacterial strains in mid-log growth phase were incubated with the homopeptides at MBC concentration for 10 to 20 min, and then the fluorescence capture was obtained.

SEM micrograph can be used for the analysis of peptide effects on bacterial membranes [52, 58–61]. Cultures of bacterial strains in mid-log growth phase were incubated with the homopeptides at MIC concentration for 20 min to 1 h. Before microscopic analysis, platinum-palladium powder was sprayed on the samples for better observation of the bacterial membrane structure. By utilizing this technique, the particular residues and structural components that wield the most significant influence in bacterial membrane disruption can be identified.

Different lipid models were used to study the interaction of the peptides with membranes [35, 50–53]. As a model, the use of lipid vesicles formulation with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phospho-(1-rac-glycerol) (DMPG), or with membrane extracts from bacterial strains were prepared by extrusion of frozen and thawed liposome suspensions through polycarbonate filters under nitrogen at 15°C above the lipid transition temperature.

Bacterial membrane extracts can be produced by bacterial pellet sonication, followed by lipid extraction by the Folch method with chloroform/methanol/water mixture. The lipids were obtained by centrifugation at 5,000 × g for 10 min, obtaining the organic phase. Samples were dried under nitrogen and resuspended in MilliQ water at 37°C in a water bath. The lipid suspension was vortexed and sonicated until the turbidity disappeared. Finally, the suspension was used to generate vesicles in the same way as with the model lipids.

A SYTOX Green uptake assay has been widely used to verify the membrane-disrupting ability of the peptides [51, 53, 57–60]. Aliquots of bacterial cell cultures in the exponential growth phase were used to prepare the samples to be read with peptide solutions at different concentrations. Controls include, on the one hand, peptides known to act as cell penetrants peptides and, on the other, membrane-disrupting peptides. The interaction and membrane disruption effects of the peptides were then monitored using a real-time thermocycler or fluorimeter. For penetrating peptides, an increase in fluorescence was not observed, whereas, for membrane-disrupting peptides, an increase in fluorescence was observed.

Hysteresis activity, using a differential scanning calorimeter implemented with an HSS8 high-sensitivity sensor, and a Huber TC45 intracooler chiller, was used to analyze the antifreezing activity of peptides [54]. Peptide solutions at 2, 10, 15, and 25 mM concentrations were used. A total of10 μL of the sample in an aluminum container was cooled down to its freezing point, from 10°C to –25°C at a rate of –3°C/min, then heated at a rate of 3°C/min to –10°C, and then slowly heated at a rate of 1°C/min until partial melting at a recorded temperature (Tho) where the ice coexisted with the peptide solution, which was determined by comparing the cooling curve (where there is recrystallization) and the subcooling curve (where there is no recrystallization). This temperature (Tho) was maintained for 10 min to enhance the interaction between the peptide and the ice crystals. The recrystallization effect of the peptide solution was observed when the temperature was lowered again to –10°C. The next step is to freeze the sample again, cooling it down to –25°C and finally heating it up to 10°C at a rate of 3°C/min to guarantee the return to the initial conditions.

1.

Antifungal peptide from hemocytes of Argopecten purpuratus, modified to enhance activity and tested against Fusarium oxysporum, Neurospora crassa, and Saprolegnia sp. [56].

After obtaining the sequences, the peptides were chemically synthesized, and their activity verified, with the exceptions of the sequences obtained from elderberry, where activity test was done with the peptide extract as a whole, and in the case of collagen where only the in silico study was carried out.

Regarding the methodology used, there are tests that are transversal, and others that are specific according to the matrix used and the purpose of the test. Below is a summary of the methods that have not been mentioned before.

Additionally, to the methods described in Chemical synthesis, there are some specific processes in the chemical synthesis for particular requirements of peptide sequences, such as cysteine-rich peptides. For example, in the case of hepcidin, which has multiple disulfide bridges, the cysteine with an orthogonal protecting group, Cys-Acm (cysteine-acetoamidomethyl), is used for each pair of cysteines in the sequence that are involved in a disulfide bridge. Four different syntheses were made for each Cys pair (Figure 5) with the goal of directing the disulfide bridge formation. After the synthesis without the final deprotection and prior to the cleavage of the peptide from the resin the Acm group is removed and the peptide is oxidized to generate the specific disulfide bond. Subsequently, the cleavage of the resin is carried out and it is expected that the other disulfide bonds will be generated naturally. With this methodology, it was determined that the most internal bridge between Cys13 and Cys14 was the one that yielded the best result.

A useful tool to determine important amino acid residues in a peptide sequence is the alanine scan, using this amino acid to mimic the removal of the side chain of each amino acid and thus its specific interactions [71–74]. Tea bag methodology for peptide synthesis is ideal for this approach, being easy to use since there is only one amino acid change per coupling in the set of peptides to be synthesized. These changes allow for verifying the importance of the residue by testing its activity and its influence on the secondary structure when analyzed by CD [52, 53, 64, 75]. One example of the sequence and its analogues by alanine scan is shown in Table 1.

It is possible to use different labels coupled to the peptides, which can be used to track their location by using techniques such as fluorescence, confocal microscopy, or flow cytometry. A summary of the common labels is shown in Table 2. These labels are useful in peptide-specific localization and mechanism of action studies.

The synthesis of very long sequences presents a methodological difficulty, since as the peptide chain grows, steric hindrance and agglomeration occur, making it difficult to couple new residues. An option for these sequences is the use of prolines as spacers, taking advantage of the characteristic of this imino acid of changing the direction of the peptide chain, thus reducing the clash between growing chains. It is also possible to use these spacers to generate coupled sequences, or bifunctional peptides. As an example of this a sequence with the capacity to bind to adipocytes and macrophages simultaneously was generated [69].

Finally, there is the possibility of using modified amino acids, to achieve some specific effect. Some examples are summarized in Table 3.