## 2 Results
### MALDI-ToF/ToFMass Spectrometry  
Following the RP-HPLC separations, the fractions with the highest purity were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) analysis, performed with a Shimadzu Axima Performance MALDI-ToF spectrometer. The samples were co-crystallized with \(\alpha\)-CHCA and further analyzed in the positive reflectron mode. The MS spectra of the synthesized peptides are shown in Figure 7.  
According to Figure 7, besides a molecular ion for each peptide ([M + H]+), sodium and potassium adducts were also noticed. Hence, Figure 7a shows two additional signals, at 546.8 Da for the \({}^{17}\)LVFF [20] adduct with Na+ and at 562.8 Da for that with the K+ ion. Regarding the conjugate with NA, the same adducts were observed at 651.6 Da for NA-\({}^{17}\)LVFF [20] with sodium and at 667.6 Da for the potassium adduct (Figure 7b). In the case of peptide 4 (\({}^{16}\)KLVF [19]), similar signals were found in the spectra (Figure 7c,d), being specific both for Na+ (at 527.6 and 632.6 Da) and K+ adducts (543.6 and 648.6 Da).  
In order to investigate the structural stability of these peptides, we utilized tandem mass spectrometry through collision-induced dissociation in a positive reflectron mode. The main purpose of this approach was to analyze the strength of the bonds between amino acids in our designed structures. Due to biomolecule activity in the human body, some proteases may cleave these peptides. Therefore, the MS/MS data could show the location of the weaker bonds and hence indicate where the cleavage may occur. The stronger the bonds, the more stable the peptide, and, as a result, it can withstand the cleavage process. Thus, the peptides could display their therapeutic effect completely. The mass spectra of the \({}^{17}\)LVFF [20] and \({}^{16}\)KLVF [19] peptides following CID, are presented in Figure 8.  
Tandem mass spectrometry of the newly designed peptides shows fascinating data. According to Figure 8a, five signals for \({}^{17}\)LVFF [20], including the molecular ion at 524.6 Da ([M + H]+), were observed. The fragmentation occurred in this particular case with the formation of y\({}^{1+}\) at 312.6 Da, b\({}^{2}\) at 360.7 Da, a\({}^{1}\) at 479.8 Da and b\({}^{3}\) at 507.8 Da. This suggests that following the CID, the weaker bonds are found between phenylalanine residues, resulting in generation of the \({}^{17}\)LV [18] fragment (the inset representation from Figure 8a).  
However, in the case of the \({}^{16}\)KLVF [19] peptide, the spectrum did not exhibit any fragmentation, even when the highest power pulsed per shot within the MALDI-ToF device was used (Figure 8b). These interesting data suggest that the peptide possesses very strong bonds, which improve biostability. Hence, with the protease activity, peptide cleavage may not occur at all. The drug based on this sequence conjugate with NA could significantly improve the neuroprotective action. Therefore, in this particular case, the peptide \({}^{16}\)KLVF [19] could attach at the A\(\beta_{(1-42)}\) monomer and fibrils, thus inhibiting fibril formation. Moreover, it could improve the \(\beta\)-sheet breaker mechanism, while with the release of NA (D), other neuroprotective functions could be improved (the inset representation from Figure 8b).  
Figure 8: The mass spectra following collision-induced dissociation in positive reflectron mode of: (a) \({}^{17}\)LVFF [20] and (b) \({}^{16}\)KLVF [19] peptides, where the orange D represent the additional drug.