Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online
Journal website www.isnff-jfb.com

Original Research

Volume 26, June 2024, pages 29-41

Substrate specificity of exopeptidases in small intestinal mucosa determines the structure of food-derived collagen peptides in rat lumen and blood

Figures

Figure 1.
Figure 1. Mass spectrometry chromatograms of compounds in collagen hydrolysate (CH) before digestion. Non-derivatized compounds were resolved by reversed-phase high-performance liquid chromatography (RP-HPLC) and detected by mass spectrometer (LC-MS/MS) in total ion scan at positive mode at different scan ranges across mass-to-charge ratio (m/z) 100–200, 200–225, 225–250, 250–275, 275–300, 300–350, 350–400, 400–500, and 500–1,000. Amino acids and peptides are indicated by a one-letter abbreviation for amino acids. O and pE represent hydroxyproline and pyroglutamic acid, respectively. *Represents the position of the Pro-Hyp (PO) peak. #Represents the peaks that could not be assigned to peptides with any combination of amino acids.
Figure 2.
Figure 2. Mass spectrometry chromatograms of AccQ derivatives in digests of collagen hydrolysate (CH). (a) In vitro digest using aminopeptidase N; (b) In vitro digest using a mixture of pancreatin, leucine aminopeptidases, and carboxypeptidases A; (c) In vitro digest using crude extract of small intestinal mucosa of rats; (d) Crude extract of mucosa blank; (e) In vitro digest using blood plasma of rats; (f) Blood plasma blank. The AccQ-derivatives were specifically detected by LC-MS/MS in precursor ion scan targeting b1 ion of AccQ moiety (m/z = 171) in positive mode across m/z range of 325–250, 350–375, 375–400, 400–450, and 450–500. #Represents the peaks that could not be assigned to peptides with any combination of amino acids.
Figure 3.
Figure 3. Peptide contents in different in vitro digests of CH. (a) CH digest using APN; (b) CH digest using mixture of pancreatin, LAP, and CPA; (c) CH digest using crude extract of small intestinal mucosa of rat; (d) CH digest with blood plasma of rat. All peptides were quantified by LC-MS/MS in multi-reaction monitoring mode (MRM). Estimated peptides were chemically synthesized and used for the optimization of MRM and external standards.
Figure 4.
Figure 4. Peptide contents in the lumen (a), small intestinal tissue (b), and blood plasma of rats (c) after ingestion of CH. The collagen group was administered CH solution at 800 mg/kg BW, and the control group with 300 µL water. After 1 h, the rats were sacrificed, and the peptides were extracted from the lumen, small intestinal tissue, and plasma. All peptides were derivatized using AccQ and quantified by LC-MS/MS in MRM mode. The results are presented as the mean ± SD. Asterisks (***), (**), and (*) represent significant differences, p < 0.001, p < 0.01, and p < 0.05, respectively, between the collagen group and control group by Studentˋs t-test (n = 3).
Figure 5.
Figure 5. Gly-Pro contents in the small intestinal lumen (a) and blood plasma (b) of rats 30 and 60 min after oral administration of synthetic Gly-Pro at a dose of 50 mg/kg body weight. The contents of stable isotope labeled Gly-Pro (G(d2)P) and Gly (G(d2)) in the small intestinal lumen (c) and blood plasma (d) at 30 minutes after oral administration at the same dose. Asterisks (**) and (*) represent significant differences, p < 0.01, and p < 0.05, respectively, between the G(d2)P30 group and control group by Studentˋs t-test (n = 3).
Figure 6.
Figure 6. Degradation of Gly-Pro and Pro-Hyp by partially purified crude extract of mucosa. Upper: Partial purification of exopeptidases in the crude extract of the mucosa by size exclusion chromatography using Superdex 75 10/300 GL column. Peak A showed the highest LAP and prolidase activities. Thus, it was selected to digest synthetic peptides. Lower: Synthetic peptides (Gly-Pro and Pro-Hyp) were digested with the partially purified mucosal enzymes. The degradation rate (%) was calculated by quantifying the liberated Pro. The results are presented as the average of duplicate analysis.

Table

Table 1. Summary of identified peptides in all digests of CH as shown in Figure 2
 
PeakPrecursor ion (m/z)MWEstimated sequenceProduct ions (m/z)
1343172Gly-Pro116.1 (y1), 171.1 (AccQ, b1), 173.2 (y2), 228.1 (b2), 343.0 (y3)
2357186Ala-Pro44.2 (*Ala), 116.1 (y1), 171.1 (AccQ, b1), 187.1 (y2), 214.2, 242.0 (b2), 357.1 (y3)
3373202Ala-Hyp132.0 (y1), 171.1 (AccQ, b1), 203.1 (y2), 242.1 (b2), 373.1 (y3)
4383212Pro-Pro70.1 (*Pro), 116.2 (y1), 171.2 (AccQ, b1), 213.0 (y2), 268.1 (b2), 383.3 (y3)
5385214Val-Pro72.0 (*Val), 116.1 (y1), 171.0 (AccQ, b1), 215.0 (y2), 242.1, 270.1 (b2), 385.2 (y3)
6387216Thr-Pro74.3 (*Thr), 116.2 (y1), 171.2 (AccQ, b1), 217.1 (y2), 244.1, 272.3 (b2), 387.1 (y3)
7399228Pro-Hyp70.1 (*Pro), 86.1 (*Hyp), 132.2 (Hyp), 171.1 (AccQ, b1), 229.2 (y2), 268.1 (b2), 399.1 (y3)
8399228Ile-Pro70.1 (*Pro), 116.2 (y1), 145.2, 171.1 (AccQ, b1), 229.1, 256.1, 284.1 (b2), 399.2 (y3)
9399228Leu-Pro70.2 (*Pro), 116.1 (y1), 145.1, 171.3 (AccQ, b1), 229.2, 256.1, 284.1 (b2), 399.1 (y3)
10401230Asp-Pro70.1 (*Pro), 116.1 (y1), 171.0 (AccQ, b1), 213.1, 231.0 (y2), 286.1 (b2)
11403232Asp-Val72.0 (*Val), 88 (*Asp), 118.1 (y1), 145.2, 171.1 (AccQ, b1), 233.1 (y2), 259.1 (a2), 286.2 (b2), 403.1 (y3)
12414243Gly-Pro-Ala70.0 (*Pro), 127.1, 155.1, 171.2 (AccQ, b1), 187.3, 200.1, 228.2 (b2), 244.2, 414.4 (y4)
13415244Glu-Pro70.3 (*Pro), 84.1, 116.2 (y1), 145.0, 171.1, 227.1, 245.0 (y2), 272.1, 300.2 (b2), 415.1 (y3)
14415244Ile-Hyp86.1 (*Ile), 132.1 (y1), 171.0 (AccQ, b1), 245.0 (y2), 256.1, 283.9 (b2), 414.9 (y3)
15415244Leu-Hyp86.1 (*Leu), 132.1 (y1), 171.0 (AccQ, b1), 245.0 (y2), 256.1, 283.9 (b2), 414.9 (y3)
16417246Leu-Asp86.2 (*Ile/Leu), 88 (*Asp), 144.6, 170.9 (AccQ, b1), 256.1, 285.2, 416.9 (y3)
17417246Met-Pro56.3, 60.8, 104 (*Met), 115.8 (y1), 170.8 (AccQ, b1), 246.9 (y2), 273.9, 301.9 (b2), 416.7 (y3)
18430259Ala-Hyp-Gly44.4 (*Ala), 86.1 (*Hyp), 144.9, 171.0 (AccQ, b1), 189.0 (y2), 213.6 (a2), 242.1 (b2), 258.6 (y3), 430 (y4)
19430259Gly-Pro-Ser70.1 (*Pro), 88.3 (*Asp), 106 (y1), 127.0, 145, 171.1 (AccQ, b1), 200.1 (a2), 203.0 (y2), 227.8 (b2), 242.0, 260.0 (y3), 268.3 (b2), 429.7 (y4)
20433262Met-Hyp55.8, 86.5 (*Hyp), 104.3 (*Met), 132.0 (y1), 170.9 (AccQ, b1), 263.1, 302.3 (b2), 433.0 (y3)
21442271Gly-Pro-Val70.2 (*Pro), 72 (*Val), 116 (y1), 117.9 (y1), 145.0, 171.0 (AccQ, b1), 200 (a2), 215.3 (y2), 228 (b2), 272.1 (y3), 442.1 (y4)
22444273Gly-Pro-Thr70.0 (*Pro), 127.0, 171.0 (AccQ, b1), 186.9, 200 (a2), 217.0 (y2), 227.9 (b2), 256.1, 273.9 (y3), 444 (y4)
23446275Ser-Hyp-Gly60.1 (*Ser), 86.1 (*Hyp), 145.2, 171.0 (AccQ, b1), 189.8 (y2), 201.1, 229.6, 258.0 (b2), 275.9, 446.3 (y4)
24449278Tyr-Pro116.1 (y1), 136.2 (*Tyr), 171.0 (AccQ, b1), 278.8 (y2), 306.2 (a2), 334.1 (b2), 448.8 (y4)
25449278Phe-Hyp120.2 (*Phe), 132.0 (y1), 171.0 (AccQ, b1), 279.1 (y2), 289.9, 318.1 (b2), 448.9 (y4)
25*450279Phe-Hyp*120.1 (*Phe), 132.3 (y1), 144.6, 171.0 (AccQ, b1), 279.9 (y2), 290.5 (a2), 317.6 (b2), 449.7 (y4)
26456285Pro-Hyp-Gly70.3 (*Pro), 86.3 (*Hyp), 141.0, 171.1 (AccQ, b1), 189.5 (y2), 286.1 (y3), 455.9 (y4)
27456285Gly-Pro-Hyp30.1 (*Gly), 70.2 (*Pro), 132.1 (y1), 171.1 (accQ, b1), 229.1 (b2, y2), 286.2 (y3), 456.1 (y4)
28460289Thr-Hyp-Gly74 (*Thr), 86.1 (*Hyp), 145.1, 171.3 (AccQ, b1), 189.1 (y2), 244, 2 (a2), 71.7 (b2), 289.8 (y3), 459.3
29472301Ile-Hyp-Gly86.5 (*Ile), 171.0 (AccQ, b1), 188.9 (y2), 255.8 (a2), 283.7 (b2), 302 (y3), 472 (y4)
30472301Leu-Hyp-Gly86.2 (*Leu), 170.8 (AccQ, b1), 188.8 (y2), 226.4, 256.1 (a2), 283.9 (b2), 302 (y3), 471.8 (y4)
31474303Gly-Pro-Met70.0 (*Pro), 127.0, 155.2, 170.8 (AccQ, b1), 200.2 (a2), 227.7 (b2), 246.5 (y2), 285.3, 304.1 (y3), 473.1
32488317Glu-Hyp-Gly76.6, 85.2 (*Hyp), 102 (*Glu), 145.3, 170.6 (AccQ, b1), 189.3 (y2), 224.8, 272.1 (a2), 300.0 (b2), 487.8 (y4)
33490319Met-Hyp-Gly55.7, 85.5 (*Hyp), 104.3 (*Met), 131.9 (y1), 170.7 (AccQ, b1), 188 (y2), 273.7, 301.6 (b2), 319.4, 489.6 (y4)
34506335Phe-Hyp-Gly86.0 (*Hyp), 119.8 (*Phe), 145.2, 170.9 (AccQ, b1), 189 (y2), 289.7, 317.6 (b2), 335.8 (y3), 505.3