## 2 Results and Discussion
### Molecular Mass Confirmation (MALDI-ToF MS and MS/MS)  
Molecular mass determination was obtained by using a MALDI-ToF MS instrument (Bruker, Billerica, MA, USA). Crystals were characterized by distinctive morphologies when the peptide was associated (co-crystalized) with HCCA and DHB matrices, as depicted  
Figure 2: HPLC chromatogram of raw FEYNF-NH\({}_{2}\) pentapeptide. The chromatogram was recorded at a wavelength of 215 nm. Injected volume: 90 \(\mu\)L.  
in Figure S3 (in Supplementary Material) and observed through the MALDI-ToF camera. Consistent with both experimental findings and existing literature, co-crystals originating from the DHB matrix exhibit a needle-like, acicular structure, while those formed from the HCCA matrix display a compact, amorphous appearance [31]. Upon mass spectrometry analysis of the first peak, the molecular ion [M + H]\({}^{+}\) of the pentapeptide was identified at \(m/z\) 718.40 with the DHB matrix (Figure 3a) and slightly shifted to \(m/z\) 718.55 with HCCA (Figure 3b). Notably, alongside the protonated molecular ion, the detection of sodium ([M + Na]\({}^{+}\)) adduct ions at \(m/z\) 740.41 and \(m/z\) 740.50, contingent upon the specific matrix employed, was evident. The peptide appears to demonstrate a distinct tendency to produce sodium ions, possibly due to interactions between the sodium ion and carboxyl oxygen and/or amide nitrogen. This interplay could achieve stability through the aromatic ring of the tyrosine side chain, a hypothesis previously supported by previous research studies [32]. Furthermore, potassium species ([M + K]\({}^{+}\)) were observed at \(m/z\) 756.51 and 756.58, as depicted in Figure 3a,b.  
Further analysis involved collision-induced dissociation (CID) of the molecular ion [FYNF + H]\({}^{+}\) (\(m/z\) 718.55), yielding multiple b\({}^{+}\) and y\({}^{+}\) molecular fragments, providing valuable insights into the fragmentation patterns of the FYNF-NH\({}_{2}\) pentapeptide and enhancing our understanding of its structural characteristics (Figure 3c). The tandem mass spectrum exhibits several distinct fragments, notably featuring ions abundant due to the elimination of ammonia: y\({}_{2}^{+}\)-NH\({}_{3}\) at \(m/z\) 261.87, y\({}_{3}^{+}\)-NH\({}_{3}\) at \(m/z\) 424.99, b\({}_{4}^{+}\)-NH\({}_{3}\) at \(m/z\) 537.99, and y\({}_{4}^{+}\)-NH\({}_{3}\) at \(m/z\) 553.61. Additionally, water loss correlates with the glutamate moiety, while carbon dioxide release likely originates from the decarboxylation of the same moiety.  
Notably, at \(m/z\) 672.93 ([M + H]\({}^{+}\)--46), the observed 46 Da discrepancy is postulated to correspond to the elimination of a CO\({}_{2}\) and a H\({}_{2}\) molecule. Moreover, scrutiny of the tandem MS spectra reveals that the equitable distribution of aromatic residues along the peptide backbone induces fragmentation at all amide bonds. This diverges from our earlier investigations, wherein the mutant peptide FESNY-NH\({}_{2}\) characterized by aromatic residues solely at the termini exhibited a marked predilection for fragmentation in those regions [33]. The inclusion of phenylalanine and tyrosine residues, endowed with a relatively high electron density, is conjectured to facilitate fragmentation by promoting charge localization and augmenting susceptibility to dissociation processes in tandem MS spectra [34].