Identiﬁcation and Characterization of Fibronectin-Binding Peptides in Gelatin

: Collagen and ﬁbronectin (FN) are important components in the extracellular matrix (ECM). Collagen-FN binding belongs to protein-protein interaction and plays a key role in regulating cell behaviors. In this study, FN-binding peptides were isolated from gelatin (degraded collagen) using afﬁnity chromatography, and the amino acid sequences were determined using HPLC-MS. The results indicated that all FN-binding peptides contained GPAG or GPPG. Matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometry (MALDI-TOF MS) and dual-polarization interferometry (DPI) were used to analyze the effects of hydroxylation polypeptide on FN binding activity. DPI analysis indicated that peptides with molecular weight (MW) between 2 kDa and 30 kDa showed higher FN-binding activity, indicating MW range played an important role in the interaction between FN and peptides. Finally, two peptides with similar sequences except for hydroxylation of prolines were synthesized. The FN-binding properties of the synthesized peptides were determined by MALDI-TOF MS. For peptide, GAPGADGP*AGAPGTP*GPQGIAGQR, hydroxylation of P8 and P15 is necessary for FN-binding. For peptide, GPPGPMGPPGLAGPPGESGR, the FN-binding process is independent of proline hydroxylation. Thus, FN-binding properties are proline-hydroxylation dependent.


Introduction
Collagen and fibronectin (FN) play a vital role in the extracellular matrix (ECM). Collagen is a biopolymer synthesized by animal cells, due to its special physiological function and high nutritional value, it is widely used in healthcare, cosmetics, and medical materials. It could prevent cardiovascular and cerebrovascular diseases and can lower blood pressure [1][2][3]. Collagen could interact with FN [4], but gelatin and denatured collagen have stronger binding activities than collagen [5]. In the ECM, the conjugation has important biological significance. The binding of collagen to FN could cause a conformational change in FN, leading to various biological responses, such as attachment to the cell surface to promote cell migration and wound repair, as well as differences in integrin receptor binding [6]. However, the absence of collagen in cultured primary fibroblasts will affect the arrangement of FN in the ECM [7]. Consequently, the study of collagen and FN binding sites is key to some relevant biological interactions.
FN, a 450 kDa glycoprotein, is composed of three modules (F1, F2, and F3). In the binding of FN and collagen, it is known that the 42 kDa fragment near the N-terminal of FN, 6F1-1F2-2F2-7F1-8F1-9F1 (n denotes the nth type in the nomenclature nFX) [8], is the collagen-binding domain. The F2 module plays a pivotal role in the interaction, but almost all the modules in the 42-kDa fragment are important [9].
In collagen, the specific FN-binding sites have not yet been identified. The binding activity is influenced by many factors, including the molecular weight (MW) and the Polymers 2022, 14, 3757 3 of 16 coupled to 3 mL of activated sepharose for 24 h at 37 • C. Active groups were blocked with ethanolamine for 4 h at 37 • C. Then, the sepharose was washed alternatively with buffer solutions at pH 8.0 and pH 4.0. The matrix was filled in the column (1.0 cm × 4.0 cm) to prepare FN-affinity column. The same method was also used to prepare the micro-FNaffinity column (100 µL).

Tryptic Digestion
Twenty milligrams of bovine gelatin were dissolved in 5 mL of ultrapure water. Two times the volume of ethanol was added, and the mixture was kept at 4 • C for 30 min. The gelatin solution was centrifuged at 10,000 r/min for 10 min to precipitate the gelatin. The high MW peptides of the precipitate were recovered in trypsin resuspension buffer (pH 8.0), and 1:50 (w/w) trypsin solution (1 µg/µL in trypsin resuspension buffer, pH 8.0) was added. The mixture was incubated at 37 • C for 12 h. [9,14,17] The digest was loaded onto the FN-affinity column and stabilized in 20 mM PBS equilibrium buffer (pH = 7.4). The column was washed in equilibrium buffer, and peptides were eluted with 3 M urea in equilibrium buffer. The eluted peptides were desalted by Sephadex G-25 (GE Healthcare, Chicago, IL, USA) and concentrated. The obtained peptides were referred to as sample 1.

FN Affinity Chromatography
The undigested gelatin was separated in the same way, and the isolated peptides were digested by trypsin at 37 • C for 12 h. The obtained peptides were referred to as sample 2.

MALDI-TOF MS Analysis
The MW ranges of samples 1 and 2 were determined using MALDI-TOF-MS (autoflex III smartbeam, Bruker, Bremen, Germany) [18]. The samples were mixed in the ratio 1:1 with the α-cyano-4-hydroxycinnamic acid matrix (5 mg/mL in 50% ACN containing 0.1% TFA), and the samples were spotted on the MALDI target. The mass scan range was set from m/z 600 to 10,000. A mass scan was performed in the positive ion mode and linear detector.
The synthesized peptides were loaded onto the micro-FN-affinity column, followed by the washing solution. The eluted peptides were also analyzed by MALDI-TOF MS.

HPLC-MS Analysis
The separated peptides in samples 1 and 2 were analyzed by HPLC-MS [19]. The HPLC-MS system consisted of an Agilent 1100 HPLC and MS (LCQ DecaXP, Thermo Electron, San Jose, CA, USA). The samples were loaded onto an Agilent Zorbax SB C18 column (2.1 mm × 150 mm, 5 µm). Mobile phase A was water (containing 0.1% TFA), and mobile phase B was ACN (containing 0.1% TFA). The HPLC gradient was 5-40% B from 0 to 50 min; 40-80% B from 50 to 80 min. The flow rate was 0.2 mL/min. The outlet of the column was introduced into the ion source of an electrospray ionization mass spectrometer. The spray voltage was 4.5 kV, and the capillary was kept at 300 • C. The data acquisition consisted of three scan events: an MS scan, followed by one zoom scan to determine the charge state of the ion, and an MS/MS scan to generate an MS/MS spectrum. The MS scan range was from m/z 400 to 2000. The zoom scan and tandem mass spectrometry (MS/MS) functions were performed in data-dependent mode. Dynamic exclusion was adopted to one count and a 0.5 min exclusion duration unit. The collision energy value was installed to 35%.

Database Searching and Data Processing
Sequence information from MS/MS data was processed using the Turbo SEQUEST algorithm in Bioworks 3.2 software (Thermo Electron, San Jose, CA, USA). A database was created by extracting bovine type I collagen entries from the Swiss-Prot/TrEMBL database (http://www.expasy.org, accessed on 15 July 2020). The database searches and SEQUEST criteria were based on a previously published method [19].

Surface Modification of DPI Chip
The unmodified DPI sensor chip (AnaChip TM , Farfield Sensors Ltd., Manchester, UK) was successively cleaned by ultrasonication in ethanol, acetone, and ultrapure water for 5 min. Then, the chip was immersed in piranha solution (7:3 sulfuric acid to hydrogen peroxide) at 90 • C for 2 h, rinsed in ultrapure water, and dried. APTES was added to anhydrous toluene to create solution concentrations of 0.001 mol/L APTES. The chip was placed in the APTES solution to react for 30 min, followed by ultrasonication in anhydrous toluene. The chip was dried by nitrogen stream. Finally, an amine-functionalized surface formed on the chip [20]. [17,20] The modified chip was installed in an AnaLight ® Bio200 dual-polarization interferometer (Farfield Sensors Ltd., Salford, UK). The laser beam (λ = 632.8 nm) with two orthogonal polarizations passed through the chip. When the protein was loaded on the chip surface, the upper sensing waveguide was changed, and the lower reference waveguide of the chip was of no influence, causing the laser to produce an evanescent field change. This resulted in a shift of the interference fringes. Subsequently, the change in transverse magnetic (TM) and transverse electric (TE) phases was used to calculate the refractive index and thickness, respectively, via the Maxwell equation. Tris-HCl buffer (pH 7.4, including the 0.1 M NaCl) flowed over the chip surface. The instrument was calibrated with ethanol and H 2 O prior to the experiment. To prepare the chip, 100 µg/mL FN was injected, followed by the sealing agent. Then, the peptides samples were injected, and the surface dimensions and densities were measured. The data were automatically analyzed by AnaLight DAQ.

Separation of Peptides through FN Affinity Chromatography
Due to the affinity of FN with gelatin, peptides in gelatin were separated by affinity chromatography, in which affinity media was prepared by coupling FN on agarose media. The sample was precipitated by ethanol and then was dissolved with PBS buffer (pH = 7.4) before adding to the separating column. The affinity chromatography results were shown in Figure 1. Fragments of nonspecific adsorption were removed by washing the column with 20 mM PBS buffer (pH 7.4), and the binding peptides were eluted with 3 M urea [21]. The elution peak with the high concentration of salt would affect the mass spectrometric detection. The peptide fraction was desalted with SephadexG-25(10 mL) ( Figure 2). Figure 1 illustrated that not all the peptides from the digested gelatin could bind to the FN, and only a few containing special sequences could bind to the N-terminal 42 kDa region of FN. We inferred that the special sequences may play an important role in FN-affinity. In Figure 2, it could be seen that the MW range of the separated peptides changed. The main peaks were distributed in 6-8 mL, which were some peptides with larger MW.

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.

MALDI-TOF MS Analysis of the MW Range
The MW range of the two separated samples was analyzed using MA The mass spectra in Figures 3 and 4 showed that the MW range of both sam approximately 1000 to less than 6000 Da but was mainly distributed betw

HPLC-MS Analysis of the Peptide Sequences
Bovine gelatin, the separated gelatin (not digested), sample 1, and sample 2 w analyzed by HPLC-MS, and the corresponding chromatograms are shown in Figure 5. similarity of the traces in Figure 5A, B indicated that gelatin contains many peptides could bind to FN. In contrast to Figure 5B-D, the retention time in Figure 5C, D was m shorter than that in Figure 5B, further indicating that the larger peptides in the gel were degraded into smaller peptides. Database searching and software analysis (2.2.6 the mass spectra shown in Figure 5C, D were performed, and the results are listed in Ta 1. Thirteen varieties of peptides were found in both samples 1 and 2. Sample 1 was tained by tryptic digestion of gelatin followed by FN affinity column separation, w

HPLC-MS Analysis of the Peptide Sequences
Bovine gelatin, the separated gelatin (not digested), sample 1, and sample 2 w analyzed by HPLC-MS, and the corresponding chromatograms are shown in Figure 5. similarity of the traces in Figure 5A, B indicated that gelatin contains many peptides t could bind to FN. In contrast to Figure 5B-D, the retention time in Figure 5C, D was m shorter than that in Figure 5B, further indicating that the larger peptides in the gela were degraded into smaller peptides. Database searching and software analysis (2.2.6 the mass spectra shown in Figure 5C, D were performed, and the results are listed in Ta 1. Thirteen varieties of peptides were found in both samples 1 and 2. Sample 1 was tained by tryptic digestion of gelatin followed by FN affinity column separation, w sample 2 was obtained by FN affinity column separation of gelation followed by try

HPLC-MS Analysis of the Peptide Sequences
Bovine gelatin, the separated gelatin (not digested), sample 1, and sample 2 were analyzed by HPLC-MS, and the corresponding chromatograms are shown in Figure 5. The similarity of the traces in Figure 5A,B indicated that gelatin contains many peptides that could bind to FN. In contrast to Figure 5B-D, the retention time in Figure 5C,D was much shorter than that in Figure 5B, further indicating that the larger peptides in the gelatin were degraded into smaller peptides. Database searching and software analysis (2.2.6) of the mass spectra shown in Figure 5C,D were performed, and the results are listed in Table 1. Thirteen varieties of peptides were found in both samples 1 and 2. Sample 1 was obtained Polymers 2022, 14, 3757 7 of 16 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 hydroxyproline 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. Ingham K.C. found that type I collagen contains at least 14 cryptic FN binding sites of similar affinity [16].
Polymers 2022, 14, x FOR PEER REVIEW and GPPG, which were repeated in one sequence. The hydroxylation modification of proline in the repeated GPAG and GPPG sequences was not high. In the report ature, the Y position of G-X-Y in the FN-binding region had lower hydroxyproline and stronger hydrophobicity, causing the collagen helix expansion to expose th FN-binding site [22]. Consequently, the active sites of FN-binding might exist in and GPPG. Ingham K.C. found that type I collagen contains at least 14 cryptic FN sites of similar affinity [16].    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 "GPPG" sequences in different peptides are highlighted in red. The specific "GPAG" sequences in different peptides are marked with underlines.
In the study by Kleinman H. K., α1-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 α1 chain and contains the vertebrate collagenase (MMP-1) cleavage site of residues 775-776. In our research, m/z 1043.31 in Table 1 was doubly charged in the zoom scan, and the ion corresponded to the sequence, GAP-GADGP*AGAPGTP*GPQGIAGQR, in the region of α1.757-780. The sequence belonged to α1-CB7 and contained the cleavage site Q-G#I-A (# represents the bond cleaved) of MMP-1 [23]. This suggested that the fragment, which independently interacted with FN in the domain of α1.757-791, could reduce to the sequence, GPQGIAGQR, in the region of α1.772-780. In addition, the sequence, GP*AG, might be another binding site. The sequence GLP*GVAGSVGEPGP*LGIAGPPGAR in the region of α2.760-783 corresponded to m/z 1058.07 (doubly charged) in Table 1, and it contained the cleavage site L-G#I-A. As a result, we inferred that in addition to the collagenase cleavage site, GPAG and GPPG were also binding sites of FN, but the excessive hydroxylation modification of the proline in the sequence GPPG weakened the adhesion to FN.
Studies showed that both α1.552-819 and 643-932 exhibit high FN-binding activity [13]. In Table 1, the peptides numbered 1-4 belonged to the region ( Figure 6). The retention time of m/z 796.8, 1042.3, 908.9, and 780.9 with two charges was 29.46, 38.34, 30.25, and 30.18 min, respectively. In addition, m/z 793, a double charge, originated from the sequence GANGAP*GIAGAP*GFP*GAR in Figure 7. The result was consistent with that reported by Fietzek et al., in which α1-CB8 and α1.124-402 could bind to FN [24]. The region of α1.220-237 shortened the binding region of α1-CB8 and made the binding site more obvious. The specific "GPPG" sequences in different peptides are highlighted in red. The specific "GPAG" sequences in different peptides are marked with underlines.
In the study by Kleinman H. K., α1-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 α1 chain and contains the vertebrate collagenase (MMP-1) cleavage site of residues 775-776. In our research, m/z 1043.31 in Table 1 was doubly charged in the zoom scan, and the ion corresponded to the sequence, GAP-GADGP*AGAPGTP*GPQGIAGQR, in the region of α1.757-780. The sequence belonged to α1-CB7 and contained the cleavage site Q-G#I-A (# represents the bond cleaved) of MMP-1 [23]. This suggested that the fragment, which independently interacted with FN in the domain of α1.757-791, could reduce to the sequence, GPQGIAGQR, in the region of α1.772-780. In addition, the sequence, GP*AG, might be another binding site. The sequence GLP*GVAGSVGEPGP*LGIAGPPGAR in the region of α2.760-783 corresponded to m/z 1058.07 (doubly charged) in Table 1, and it contained the cleavage site L-G#I-A. As a result, we inferred that in addition to the collagenase cleavage site, GPAG and GPPG were also binding sites of FN, but the excessive hydroxylation modification of the proline in the sequence GPPG weakened the adhesion to FN.
Studies showed that both α1.552-819 and 643-932 exhibit high FN-binding activity [13]. In Table 1, the peptides numbered 1-4 belonged to the region ( Figure 6). The retention time of m/z 796.8, 1042.3, 908.9, and 780.9 with two charges was 29.46, 38.34, 30.25, and 30.18 min, respectively. In addition, m/z 793, a double charge, originated from the sequence GANGAP*GIAGAP*GFP*GAR in Figure 7. The result was consistent with that reported by Fietzek et al., in which α1-CB8 and α1.124-402 could bind to FN [24]. The region of α1.220-237 shortened the binding region of α1-CB8 and made the binding site more obvious.  Our research identified some binding peptides from the α2 chain. Guidry C. digested collagen I and II using CNBr and found that both CB4 from α2(I) homologous with CB10 from α1(II) and CB5 from α2(I) homologous with CB12 from α1(II) exhibited FN-binding activity. CB4 and CB5 were located in the regions of α2(I).0-300 and 693-1011, but the binding activity of CB4 was weaker than that of CB5 [15]. Table 1 showed that the peptides of regions α2.102-111, α2.708-723, α2.856-873, and α2.931-941 were included in the binding region. This further suggested that the bound region could cut down these sequences. They contained the same peptide sequences of GPAG and GPPG. Therefore, GPAG or GPPG was the binding site of FN. Moreover, this is the first report on these sequences in α1.91-99, α2.328-341, α2.451-465, α2.469-486, and α2.672-692 (Table 1)

DPI Analysis of the Effect of MW on FN Binding
After FN was immobilized on the chip, the 1, 3, and 10 kDa peptides and bovine gelatin were, respectively, injected into the two channels of DPI and reacted with immobilized FN. The MW range of bovine gelatin was 1.4-33 kDa, as determined by gel filtration chromatography. The adsorption conditions of these samples were shown in Figures  9-11, including the real-time profile (A) and the resolved layer thickness-mass (B). Our research identified some binding peptides from the α2 chain. Guidry C. digested collagen I and II using CNBr and found that both CB4 from α2(I) homologous with CB10 from α1(II) and CB5 from α2(I) homologous with CB12 from α1(II) exhibited FNbinding activity. CB4 and CB5 were located in the regions of α2(I).0-300 and 693-1011, but the binding activity of CB4 was weaker than that of CB5 [15]. Table 1 showed that the peptides of regions α2.102-111, α2.708-723, α2.856-873, and α2.931-941 were included in the binding region. This further suggested that the bound region could cut down these sequences. They contained the same peptide sequences of GPAG and GPPG. Therefore, GPAG or GPPG was the binding site of FN. Moreover, this is the first report on these sequences in α1.91-99, α2.328-341, α2.451-465, α2.469-486, and α2.672-692 (Table 1) and their binding with FN. For example, the sequence GFSGLDGAK corresponded to m/z 851.4, and the matched b and y ions were shown in Figure 8. Our research identified some binding peptides from the α2 chain. Guidry C. digested collagen I and II using CNBr and found that both CB4 from α2(I) homologous with CB10 from α1(II) and CB5 from α2(I) homologous with CB12 from α1(II) exhibited FN-binding activity. CB4 and CB5 were located in the regions of α2(I).0-300 and 693-1011, but the binding activity of CB4 was weaker than that of CB5 [15]. Table 1 showed that the peptides of regions α2.102-111, α2.708-723, α2.856-873, and α2.931-941 were included in the binding region. This further suggested that the bound region could cut down these sequences. They contained the same peptide sequences of GPAG and GPPG. Therefore, GPAG or GPPG was the binding site of FN. Moreover, this is the first report on these sequences in α1.91-99, α2.328-341, α2.451-465, α2.469-486, and α2.672-692 (Table 1) and their binding with FN. For example, the sequence GFSGLDGAK corresponded to m/z 851.4, and the matched b and y ions were shown in Figure 8.

DPI Analysis of the Effect of MW on FN Binding
After FN was immobilized on the chip, the 1, 3, and 10 kDa peptides and bovine gelatin were, respectively, injected into the two channels of DPI and reacted with immobilized FN. The MW range of bovine gelatin was 1.4-33 kDa, as determined by gel filtration chromatography. The adsorption conditions of these samples were shown in Figures  9-11, including the real-time profile (A) and the resolved layer thickness-mass (B).

DPI Analysis of the Effect of MW on FN Binding
After FN was immobilized on the chip, the 1, 3, and 10 kDa peptides and bovine gelatin were, respectively, injected into the two channels of DPI and reacted with immobilized FN. The MW range of bovine gelatin was 1.4-33 kDa, as determined by gel filtration chromatography. The adsorption conditions of these samples were shown in Figures 9-11, including the real-time profile (A) and the resolved layer thickness-mass (B).  In Figure 9B, the thickness increased from 11.60 nm to 12.55 nm, and the mass increased from 3.68 to 3.86 ng/mm 2 . The obvious increases in thickness and mass demonstrated that gelatin of MW range 1.4-33 kDa interacted with the immobilized FN. Certainly, not the entirety of the MW range (1.4-33 kDa) could bind to FN; thus, 1, 3, and 10 kDa peptides were further analyzed.  According to DPI data analysis, the 1 kDa sample failed to bind FN (Figure not shown). Although the thickness did not increase significantly, there was a significant increase in the mass in the 3 kDa sample (Figure 10). This proved that the 3 kDa sample bound to FN.
Similarly, the mass and thickness of the 10 kDa sample indicated that it could interact with FN ( Figure 11).  In Figure 9B, the thickness increased from 11.60 nm to 12.55 nm, and the mass increased from 3.68 to 3.86 ng/mm 2 . The obvious increases in thickness and mass demonstrated that gelatin of MW range 1.4-33 kDa interacted with the immobilized FN. Certainly, not the entirety of the MW range (1.4-33 kDa) could bind to FN; thus, 1, 3, and 10 kDa peptides were further analyzed. It is known that the FN-binding activity of collagen is lower than that of gelatin [5]. Our study suggested that the peptides lower than 1 kDa did not readily bind to FN. Thus, the bigger MW of peptides, the better the FN-binding activity. In other words, there was stronger binding activity in the suitable MW range of peptides, because a modest MW might radically expose more FN-binding sites than collagen. In this paper, peptides in the range of 2-30 kDa exhibited better affinity to FN. Therefore, the MW plays an important role in the interaction between FN and peptides.

MALDI-TOF MS Analysis of the Effect of Hydroxylation Modification on FN Binding
The sequence and MW of the four synthesized peptides were listed in Table 2. They were the peptides of FN-binding in Table 1 and contained two non-hydroxylation (1 and 3) and two hydroxylation modifications (2 and 4). In order to investigate the effect of hydroxylation modification on FN binding, peptides 1 and 2, and peptides 3 and 4, were, respectively, mixed in a 1:1 mass ratio and separately named GIA and GPP. The two mixed samples were separately loaded onto the micro affinity column. The column was eluted until the washing solution did not include the sample by MALDI-TOF MS, following the elution solution analyzed. Figures 12 and 13 showed the MALDI-TOF mass spectra of the two samples' elution. range of 2-30 kDa exhibited better affinity to FN. Therefore, the MW plays an importan role in the interaction between FN and peptides.

MALDI-TOF MS Analysis of the Effect of Hydroxylation Modification on FN Binding
The sequence and MW of the four synthesized peptides were listed in Table 2. The were the peptides of FN-binding in Table 1 and contained two non-hydroxylation (1 an 3) and two hydroxylation modifications (2 and 4). In order to investigate the effect of hy droxylation modification on FN binding, peptides 1 and 2, and peptides 3 and 4, wer respectively, mixed in a 1:1 mass ratio and separately named GIA and GPP. The tw mixed samples were separately loaded onto the micro affinity column. The column wa eluted until the washing solution did not include the sample by MALDI-TOF MS, follow ing the elution solution analyzed. Figures 12 and 13 showed the MALDI-TOF mass spectr of the two samples' elution.   In Figure 12A, m/z 2056.01 and 2088.30 were produced by peptides 1 and 2 from Table 2, respectively, and m/z 2078.19 was m/z 2056.01 + Na + . The figure showed that the washing solution no longer included the GIA sample after the column was fully washed (Figure not shown). By comparing Figure 12A,B, the larger peak area of peptide 1 developed into smaller than that of peptide 2. The differences between the spectra indicated that the hydroxylation modification of peptide 2 led to stronger binding activity than the nonhydroxylation of peptide 1. Again, the appropriate position and amount of hydroxyproline in the sequence of peptide 2 had a significant influence on the binding activity.
In comparison with Figure 13A, the peak areas in Figure 13B did not significantly change. We hypothesized that the amount of proline in the sequence of peptide 3 was so high that slight hydroxylation modifications might influence the binding, or even inhibit it.
Interestingly, all four synthesized peptides could interact with FN, and it was incorrect to infer that only the sequences with hydroxylation modification could bind to FN. The addition of the binding was also attributed to the position and amount of hydroxyproline, both of which are essential. In Figure 12A, m/z 2056.01 and 2088.30 were produced by peptides 1 and 2 from T 2, respectively, and m/z 2078.19 was m/z 2056.01 + Na + . The figure showed that the was solution no longer included the GIA sample after the column was fully washed (Fi not shown). By comparing Figure 12A,B, the larger peak area of peptide 1 developed smaller than that of peptide 2. The differences between the spectra indicated that the droxylation modification of peptide 2 led to stronger binding activity than the non droxylation of peptide 1. Again, the appropriate position and amount of hydroxypro in the sequence of peptide 2 had a significant influence on the binding activity.
In comparison with Figure 13A, the peak areas in Figure 13B did not significa change. We hypothesized that the amount of proline in the sequence of peptide 3 wa high that slight hydroxylation modifications might influence the binding, or even in it.
Interestingly, all four synthesized peptides could interact with FN, and it was in rect to infer that only the sequences with hydroxylation modification could bind to The addition of the binding was also attributed to the position and amount of hydr proline, both of which are essential.

Conclusions
Affinity chromatography was used to isolate peptides that could bind to FN, and amino acid sequence of peptides was determined by HPLC-MS. MALDI-TOF MS and were used to analyze the effects of MW and hydroxylation modification of peptide FN binding activity.
The sequences of peptides that could bind FN all contained GPAG or GPPG; t they may be regarded as the core sequences of FN binding peptides. The MW of t peptides was mainly distributed in the range of 1000-2000 Da, and the FN-binding reg in peptide sequences were identified. These peptides, with MW between 1000 and Da, are also more easily absorbed by the human body, providing a foundation for development of new functional health products.

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 of analysis, as well as a theoretical basis for studying the binding characteristics of peptides to FN.