# Identification and Characterization of Fibronectin-Binding Peptides in Gelatin
## 3 Results and Discussion
### MALDI-TOF MS Analysis of the MW Range  
The MW range of the two separated samples was analyzed using MALDI-TOF MS. The mass spectra in Figures 3 and 4 showed that the MW range of both samples was from approximately 1000 to less than 6000 Da but was mainly distributed between 2000 and 3000 Da. In Figure 3, the analysis for sample 1 indicated that the separated peptides were degraded into the lower MW range, and the sequences could be analyzed by HPLC-MS. The analysis of sample 2 in Figure 4 indicated that the lower MW peptides of the digested gelatin could also bind to FN alone. Figure 1: Affinity chromatography of separation peptides in the gelatin. Figure 2: Gel filtration chromatograms of the desalinated peptide fraction. by tryptic digestion of gelatin followed by FN affinity column separation, while sample 2 was obtained by FN affinity column separation of gelation followed by tryptic digestion. The identified peptides in sample 2 consisted of shorter sequences that could directly bind to FN, and so do the extended sequences before tryptic digestion. Therefore, the region of the FN-binding sites could be narrowed to these sequences, including GPAG and GPPG, which were repeated in one sequence. The hydroxylation modification degree of proline in the repeated GPAG and GPPG sequences was not high. In the reported literature, the Y position of G-X-Y in the FN-binding region had lower hydroxproline content and stronger hydrophobicity, causing the collagen helix expansion to expose the active FN-binding site [22]. Consequently, the active sites of FN-binding might exist in GPAG and GPPG. [PERSON] found that type I collagen contains at least 14 cryptic FN binding sites of similar affinity [16]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Number** & \(\mathbf{m}\mathbf{z}\) & **Position** & **Sequence** \\ \hline
1 & 1590.82 & \(\alpha\)1.586–603 & GLTGPGPGP*GPAGAP*GDK \\
2 & 2089.02 & \(\alpha\)1.757–780 & GAPGADGP*AGAGGTG*GPGIAGQR \\
3 & 1816.88 & \(\alpha\)1.817–836 & GPP*GPGPFLAGPP*GESGR \\
4 & 1560.81 & \(\alpha\)1.889–906 & GETGPAGAGPGIGPVGAR \\
5 & 868.47 & \(\alpha\)2.102–111 & VGAP*GPAGAR \\
6 & 1287.64 & \(\alpha\)2.328–41 & GFP*GP*GNIGPAGK \\
7 & 1427.74 & \(\alpha\)2.451–465 & GTP*GFGFGF*AGAR \\
8 & 1580.77 & \(\alpha\)2.469–486 & GPP*GESGAGPTTG!GSR \\
9 & 1845.92 & \(\alpha\)2.672–692 & TGPP*GP*SGISGPP*GPGPGP*AGK \\
10 & 1492.70 & \(\alpha\)2.708–723 & SGETGASGPP*GFVGEK \\
11 & 2115.13 & \(\alpha\)2.760–783 & GLP*GVAGSVGEGFP*LIGAPPGR \\
12 & 1580.79 & \(\alpha\)2.856–873 & GEP*GP*AGAVGP*AGAVGP*R \\
13 & 895.46 & \(\alpha\)2.931–941 & GPAGPSGPAGK \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Peptides in samples 1 and 2 identified from database searching. Figure 5: Total ion chromatograms of the bovine gelatins digested at 37 \({}^{\circ}\)C for 12 h. (**A**) Bovine gelatin; (**B**) separated gelatin before sample 1 was digested; (**C**) sample 1; (**D**) sample 2. Then, the sequence RGEP*GPP*GPAG (*indicated the hydroxylation modification of the proline) and GAPG (n = 39, n represents the number of repeats in the chain), GPAG (n = 61), GARG (n = 19), and GERG (n =19) were synthesized, but they failed to bind FN. The moderate hydroxylation modification of proline might play an important role in recognition by FN. Furthermore, the shorter synthesized peptides, which contained FN-binding sites, failed to bind FN, indicating that the combined action of other residues was clearly required for full affinity. The specific \"GPP* sequences in different peptides are highlighted in red. The specific \"GPAG\" sequences in different peptides are marked with underlines. In the study by [PERSON], \(\alpha\)1-CB7, a type I collagen, was digested with cyanogen bromide and was identified as the classic fragment of the FN-binding [12]. The fragment lies within residues 757-791 of the \(\alpha\)1 chain and contains the vertebrate collagenase (MMP-1) cleavage site of residues 775-776.