# Identification and Characterization of Fibronectin-Binding Peptides in Gelatin
## 4 Conclusions  
Affinity chromatography was used to isolate peptides that could bind to FN, and the amino acid sequence of peptides was determined by HPLC-MS. MALDI-TOF MS and DPI were used to analyze the effects of MW and hydroxylation modification of peptides on FN binding activity.  
The sequences of peptides that could bind FN all contained GPAG or GPPG; thus, they may be regarded as the core sequences of FN binding peptides. The MW of these peptides was mainly distributed in the range of 1000-2000 Da, and the FN-binding regions in peptide sequences were identified. These peptides, with MW between 1000 and 2000 Da, are also more easily absorbed by the human body, providing a foundation for the development of new functional health products.  
DPI was used to measure the adsorption thickness and mass of peptides with different MW ranges on a chip with immobilized FN. Peptides with MW below 1 kDa did not readily bind to FN, while peptides with MW between 2 and 30 kDa were more likely to bind to FN. The result indicates that peptides with moderate MW exert greater binding activity, where more FN binding sites are exposed in the peptide sequence. Therefore, the MW range plays an important role in the interaction between FN and peptides.  
Two different sequences of peptides (including hydroxylated and unmodified peptides) were synthesized, and the amount of hydroxylated and unhydroxylated peptides was mixed. MALDI-TOF MS was used to measure the influence of hydroxylated modification on FN binding, and all four synthetic polypeptides could bind to FN. However, the location and amount of hydroxylation modification play a key role in binding strength.  
In this paper, bio-enzymatic hydrolysis technology was used to degrade gelatin, and combined with mass spectrometry analysis, the core sequence of FN binding in peptides was identified at the molecular level. The FN binding domain of the peptide sequence was shortened, providing a strategy for new raw materials and a basis for the study of functional factors of functional food. In this paper, DPI and MALDI-TOF MS were used for the first time to analyze the dynamic binding process of peptides to FN online, and the influence of various factors on the binding was discussed. The results provide a new technical method  
Figure 13: MALDI-TOF MS spectra of (**A**) GPP sample and (**B**) the elution solution.  
of analysis, as well as a theoretical basis for studying the binding characteristics of peptides to FN.  
Conceptualization, G.Z.; methodology, G.Z., J.G., L.L., J.K. and Y.K.; formal analysis, J.G., L.L. and Y.L.; writing--original draft preparation, L.L. and Y.L.; writing--review and editing, Y.L.; supervision, G.Z. and X.L. All authors have read and agreed to the published version of the manuscript.  
This work was supported by the National Key Technology R&D Programs of China (Grant No. 2021 YFC2400804), the Open Funding Project of the State Key Laboratory of Biochemical Engineering (Grant No. 2021 KF-04), and the Independent Research Project of the State Key Laboratory of Biochemical Engineering (Grant No. 2021 ZZ-03).  
Not applicable.  
The data presented in this study are available on request from the corresponding author.  
The authors declare no conflict of interest.