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Data in Brief logoLink to Data in Brief
. 2022 Jul 28;43:108492. doi: 10.1016/j.dib.2022.108492

Digestomic data of proteolysis during whether post rumen digestion after tannin supplementation

Christophe Chambon a,, Thierry Sayd b, Sylvie Bourillon b, Laetitia Theron b, Vincent Niderkorn c
PMCID: PMC9343927  PMID: 35928346

Abstract

The protein degradation of alfalfa hay after tannin supplementation was monitored during wethers digestion. Three rumen-cannulated wethers were infused a tannin solution, and water for control, through the cannula. The digestion time-points samples were collected in vivo in the rumen and in vitro in the abomasum, and the small intestine compartments. The digestomic dataset was acquired by identifying and quantifying the peptides resulting from the protein degradation, using high-resolution LC-MS/MS mass spectrometry and label-free quantitation. The digestomic dataset is the compilation of proteomic data acquired in the rumen and peptidomic data acquired in the abomasum and in the small intestine. The proteomic analysis identified 20 Medicago proteins in the rumen fluid, based on 169 peptides of which 140 are unique. The peptidomic analysis identified 28 Medicago proteins in the abomasum, based on 575 peptides of which 363 are unique, and 11 Medicago proteins in the small intestine, based on 94 peptides of which 63 are unique. This digestomic dataset of proteolysis during sheep post rumen digestion after tannin supplementation reveals the protein regions protected by tannin supplementation, and could be reused in studies related to the protein use efficiency by ruminants.

Keywords: Digestomic, Rumen, Proteomic data, Peptidomic data, Dynamic in vitro digestion, Mass spectrometry

Specifications Table

Subject Agricultural science
Specific subject area Analysis LC-MS/MS of ruminal digestion products obtained from rumen and a dynamic in vitro digestive system (DIDGI® (abomasum/small intestine)).
Type of data Figure, Tables
How the data were acquired NanoLC-MS/MS (Ultimate 3000 nanoRSLC-QExactive HF-X or LTQ-Orbitrap mass spectrometer (Thermo-Fisher Scientific, Villebon-sur-Yvette, France)
Data format LC-MS/MS Raw data (Thermo-Fisher Scientific, Villebon-sur-Yvette, France) Identification and quantitation data (ProgenesisQI, Nonlinear Dynamics, Waters, Newcastle, UK)
Description of data collection The mass spectrometry data were recorded with Xcalibur, the mass spectrometer control software. These raw data (.raw) were transformed into reprocessed data (.MZNLD) by the ProgenesisQI quantitation software (Nonlinear Dynamics). The quantified peptides and proteins identified by MASCOT (Matrix Science) and/or Peaks (Bioinformatics Solutions Inc) were analyzed by statistical tools integrated in ProgenesisQI, to give information on the hydrolysis of proteins in each studied compartment. Normalization was applied using a scalar factor on each sample to recalibrate them to a reference run.
Data source location Institution: INRAE
City/Town/Region: Saint-Genès-Champanelle
Country: France
Data accessibility All additional data provided in this article are open access by downloading Excel files:Repository name: Pride project via ProteomeXchange Data identification number: PXD032973, Direct URL to data: https://www.ebi.ac.uk/pride/archive/projects/PXD032973
Related research article T. Sayd, C. Chambon, M. Popova, D. P. Morgavi, A. Torrent, S. Blinet, L. Théron, V. Niderkorn. Impact of tannin supplementation on proteolysis during post-ruminal digestion in wethers using a dynamic in vitro system: a plant (Medicago sativa) digestomic approach. Journal of Agricultural and Food Chemistry, 2022, 70, 2221-2230 (https://doi.org/10.1021/acs.jafc.1c07378)

Value of the Data

  • The data provide the peptides and proteins identified during sheep digestion in the rumen, and in simulated abomasum and small intestine after tannin supplementation.

  • Protein and peptide sequences protected by tannin during sheep digestion are identified and quantified in rumen, abomasum and small intestine.

  • This peptidomic and proteomic datasets provide new insights into the animal nutrition and feeding efficiency in ruminants and give new elements to optimise feed supplementation and significantly decrease environmental impact through nitrogen release.

  • The digestomic dataset presented can be used to compare with in vivo digestion data to further consider this approach as an alternative to animal experiment according to the ‘R’ principles.

1. Data Description

1.1. Rumen Proteomic

In Rumen, 140 unique peptides were identified from the digestion of 20 medicago proteins with at least two unique peptides (Table 1a and via the PRIDE repository with the dataset identifier PXD032973). The majority of the identified peptides were derived from the 5 most represented medicago sativa proteins. These unique peptides (or peptides without sequence conflicts) for each protein were used for label free quantitation (Table 1b and all the individual raw data acquired during the digestion kinetic in both Control and Tannin groups are available via the PRIDE repository with the dataset identifier PXD032973, https://www.ebi.ac.uk/pride/archive/projects/PXD032973).

Table 1a.

Proteomic list of proteins identified in the rumen compartment during wether digestion of alfalfa hay supplemented in tannins.

Protein name Mass Accession number Gene name Peptide count Unique peptides Confidence score
Histone H3.2, partial 14361.8 AAB36495 H3 3 3 79.96
Chlorophyll a/b binding protein 28312.3 AAC25775 CARCAB1 7 7 365.50
Glycolate oxidase, partial 30550.2 AAC32392 GOX 4 4 216.68
Rubisco activase, partial 30018.3 AAN15946 Rca 6 6 420.07
Heat shock protein 70 70996.5 AAV98051 HSP70-1 10 9 674.11
Pentameric polyubiquitin, partial 21187.5 AAZ32851 UBQ11 4 2 164.96
Fructose bisphosphate aldolase 42963.9 ACP40514 FBA 5 5 303.42
Putative glyceraldehyde-3-phosphate dehydrogenase 43000.0 ACV32597 GADPH 13 9 710.20
ATP1, partial 37426.0 ADL63246 atp1 4 2 157.95
Photosystem I P700 chlorophyll a apoprotein A1 83295.9 AMC31418 psaA 2 2 104.76
Ribulose-1,5-bisphosphate carboxylase/oxygenase 52626.9 AMC32005 rbcL 50 37 3952.36
Apocytochrome f 35948.9 AWW91860 petA 7 7 370.41
Rab protein 24012.1 CAA55865 Rab 5 5 138.28
Ferritin 28078.8 CAA65771 FER 2 2 55.43
Ribulose bisphosphate carboxylase small chain, chloroplastic 20251.2 O65194 RBCS 12 11 900.15
Glutamine synthetase leaf isozyme, chloroplastic 47115.5 Q9XQ94 GS2 2 2 42.93
Actin-7 41711.8 XP_003602545 actin 11 11 839.94
Photosystem I P700 chlorophyll a apoprotein A2 82427.3 YP_001381735 psaB 3 3 120.18
ATP synthase CF1 beta subunit 52746.5 YP_009141595 atpB 13 9 747.17
ATP synthase CF1 alpha subunit 55691.8 YP_009141617 atpA 6 4 431.89

For each identified protein, the protein name, accession number, gene name, and confidence score were given by MASCOT or Peaks search engines. Mass is the protein molecular weight in Da. Peptide count is the number of peptides used for identification. Unique peptides are the number of peptides belonging to only one protein, and used for quantitation.

Table 1b.

Proteomic list of proteins quantified in the rumen compartment during wether digestion of alfalfa hay supplemented in tannins.

Condition Tannin
Condition control
Description T4 T5 T6 C1 C2 C3 Anova (p) q Value
Histone H3.2, partial 292999.1 213174.7 200996.4 267962.4 2720471.3 218560.1 0.358 0.337
Chlorophyll a/b binding protein 544875.2 258075.3 632179.4 480376.6 1884755.2 554884.3 0.325 0.322
Glycolate oxidase, partial 314787.0 242773.8 230646.0 28023.1 310523.3 113454.8 0.244 0.285
Rubisco activase, partial 1148583.1 626703.3 614768.3 146612.0 569681.5 217502.5 0.078 0.181
Heat shock protein 70 635331.7 486510.4 411899.0 1740900.1 9479203.8 2562796.2 0.021 0.119
Pentameric polyubiquitin, partial 1649985.1 903976.0 974430.7 2186301.1 10718614.6 5000967.9 0.042 0.132
Fructose bisphosphate aldolase 666929.8 495986.4 493078.6 328746.5 1392891.8 1204722.9 0.436 0.357
Putative glyceraldehyde-3-phosphate dehydrogenase 1121232.6 502703.1 536623.8 145669.6 2062278.1 382199.2 0.713 0.442
ATP1, partial 45306.3 65751.2 30848.5 10737.8 158880.0 127772.6 0.763 0.458
Photosystem I P700 chlorophyll a apoprotein A1 41780.5 21835.3 66875.6 61938.3 220868.5 135880.4 0.082 0.181
Ribulose-1,5-bisphosphate carboxylase/oxygenase 36565782.0 14377637.4 11799474.5 905957.3 6623227.4 2836924.7 0.043 0.133
Apocytochrome f 331301.9 230810.8 367722.6 274847.2 2150485.8 468034.8 0.295 0.318
Rab protein 177677.8 106270.1 94575.1 275243.8 1225792.7 479982.5 0.035 0.129
Ferritin 86707.1 65120.6 43724.1 42948.7 299393.6 132846.9 0.341 0.33
Ribulose bisphosphate carboxylase small chain, chloroplastic 18837450.9 10471723.5 8064851.8 996454.8 4789446.7 2368272.8 0.034 0.129
Glutamine synthetase leaf isozyme, chloroplastic 59995.0 34972.1 30827.6 1723.9 16568.4 8287.3 0.056 0.147
Actin-7 4935346.8 4541437.5 3165078.9 1219273.4 12512883.4 7159569.7 0.85 0.475
Photosystem I P700 chlorophyll a apoprotein A2 41428.5 18559.8 53498.3 41685.5 170371.5 30359.5 0.422 0.355
ATP synthase CF1 beta subunit 661058.2 547722.0 414574.4 66131.4 428958.7 755947.1 0.435 0.357
ATP synthase CF1 alpha subunit 647667.5 426021.6 358597.3 82394.4 695369.1 317001.5 0.432 0.357

For each quantified protein, the normalized abundances are given in the three individuals of each comparative group, i.e. ‘Tannin’ (T4, T5, and T6) and ‘Control’ (C1, C2, and C3). The Anova p-value and the power q-value are the results of the quantitative analysis performed using ProgenesisQI software.

1.2. Abomasum Peptidomics

In the abomasum, 363 unique peptides were identified from the digestion of 28 medicago proteins with at least two unique peptides (Table 2a and via the PRIDE repository with the dataset identifier PXD032973). Such as for the rumen proteomic results, the 5 most represented proteins in number of identified peptides account for about 60% of the identified peptides in this digestive compartment. These unique peptides for each protein were used for label-free quantitation (Tables 2b, c and all the individual raw data acquired during the digestion kinetic in both Control and Tannin groups are available via the PRIDE repository with the dataset identifier PXD032973(https://www.ebi.ac.uk/pride/archive/projects/PXD032973).

Table 2a.

Peptidomic list of proteins identified in the abomasal compartment during wether digestion of alfalfa hay supplemented in tannins.

Description Mass Accession Gene Peptide count Unique peptides Confidence score
Aquaporin-like transmembrane channel protein 31230.0 AAB86380 pAFI 8-1 6 4 340.28
Malate dehydrogenase precursor 38397.0 AAB99754 gmdh 2 2 131.57
Chlorophyll a/b binding protein 28351.0 AAC25775 CARCAB1 51 51 3229.86
Glycolate oxidase, partial 30588.0 AAC32392 GOX 6 6 367.63
Rubisco activase, partial 30170.0 AAN15946 Rca 13 13 691.57
Ferredoxin-dependent glutamate synthase, partial 26791.5.0 AAT38954 gltB_C 2 2 89.76
Heat shock protein 70 71351.0 AAV98051 HSP70-1 4 2 215.29
Pentameric polyubiquitin, partial 21187.5 AAZ32851 UBQ 4 4 287.39
Fructose bisphosphate aldolase 43165.0 ACP40514 FBA 9 9 455.95
Putative glyceraldehyde-3-phosphate dehydrogenase 43258.0 ACV32597 GADPH 16 14 990.83
Photosystem I P700 chlorophyll a apoprotein A1 83471.0 AMC31418 psaA 39 38 2018.76
Ribulose-1,5-bisphosphate carboxylase/oxygenase 52993.0 AMC32005 rbcL 205 10 13889.10
Apocytochrome f 36040.0 AWW91860 petA 17 17 1178.71
Photosystem II CP43 chlorophyll apoprotein 50482.0 AWW91876 psbC 5 5 240.01
Histone H3, partial 13915.0 CAA05554 H3-1.1 6 2 270.11
Ferritin 28061.0 CAA65771 FER 2 2 100.18
Photosystem II protein D2 39738.0 NP_054491 psbD 12 12 666.78
Ribulose bisphosphate carboxylase small chain, chloroplastic 20251.2 O65194 RBCS 33 32 2142.95
Photosystem II protein D1 39111.0 P04998 psbA 15 14 905.08
Probable aquaporin TIP-type 25324.4 P42067 MCP1 5 5 322.39
Adenosylhomocysteinase 53744.0 P50246 SAHH 2 2 69.84
Actin-7 41913.0 XP_003602545 actin2 10 9 572.66
Photosystem II 47 kDa protein 56131.0 YP_001381703 psbB 22 22 1360.88
Photosystem II protein V 9400.6 YP_001381711 psbE 2 2 85.46
Photosystem I P700 chlorophyll a apoprotein A2 82489.0 YP_001381735 psaB 45 45 2523.04
Cytochrome b6 24112.5 YP_002149762 petB 2 2 81.96
ATP synthase CF1 beta subunit 52771.0 YP_009141595 atpB 24 21 1271.26
ATP synthase CF1 alpha subunit 55714.0 YP_009141617 atpA 16 16 982.22

For each identified protein, the protein name, accession number, gene name, and confidence score were given by MASCOT or Peaks search engines. Mass is the protein molecular weight in Da. Peptide count is the number of peptides used for identification. Unique peptides are the number of peptides belonging to only one protein, and used for quantitation.

Table 2b.

Peptidomic list of proteins quantified in the abomasal compartment at 15 minutes of wether digestion of alfalfa hay supplemented in tannins.

Condition Control
Condition Tannin
Description T4 T5 T6 C1 C2 C3 Anova (p value) q value
Aquaporin-like transmembrane channel protein 1857.3 14873.6 15050.0 73803.0 99424.6 103865.7 0,001 0,002
Malate dehydrogenase precursor 15708.2 83843.4 36613.2 11600.8 16443.0 10775.8 0,001 0,002
Chlorophyll a/b binding protein 5562923.7 2930411.5 3382917.8 5348125.7 5878944.7 7575674.6 0,003 0,003
Glycolate oxidase, partial 442933.8 459805.7 331436.1 90200.2 99275.9 64640.6 0,000 0,000
Rubisco activase, partial 438692.8 813921.2 392757.9 412509.3 535492.9 354148.6 0,101 0,045
Ferredoxin-dependent glutamate synthase, partial 26890.0 64330.0 8489.0 2342.0 15645.9 5215.6 0,003 0,002
Heat shock protein 70 35482.2 36485.2 10186.0 44275.0 38132.3 29724.0 0,158 0,066
Pentameric polyubiquitin, partial 368210.6 590896.4 215780.0 331694.7 269554.2 200907.0 0,106 0,046
Fructose bisphosphate aldolase 342186.0 716798.5 201459.4 176814.6 189979.4 150231.5 0,015 0,010
Putative glyceraldehyde-3-phosphate dehydrogenase 461655.7 932166.5 221701.3 414052.7 569441.1 349280.3 0,097 0,045
Photosystem I P700 chlorophyll a apoprotein A1 571742.2 397239.6 704973.5 855340.1 1106649.5 1323653.0 0,000 0,001
Ribulose-1,5-bisphosphate carboxylase/oxygenase 1722084.7 3338112.2 3658436.2 317144.0 301861.9 209271.9 0,000 0,000
Apocytochrome f 749718.4 853491.8 1483049.0 2039160.4 2526285.3 2263769.7 0,001 0,002
Photosystem II CP43 chlorophyll apoprotein 169847.6 47204.8 203904.8 158969.3 230529.1 235333.9 0,022 0,013
Histone H3, partial 64440.4 126107.5 41682.6 68736.8 92847.9 46001.0 0,453 0,148
Ferritin 107706.4 16375.3 122897.2 25078.1 29882.0 16768.7 0,196 0,075
Photosystem II protein D2 360817.8 419712.0 478604.4 789312.5 948430.9 1086873.6 0,000 0,001
Ribulose bisphosphate carboxylase small chain, chloroplastic 2779311.7 4877198.0 5256231.9 1296395.7 1315805.3 1001594.3 0,000 0,000
Photosystem II protein D1 322122.5 171776.1 295420.7 343115.8 459884.7 526318.9 0,001 0,001
Probable aquaporin TIP-type 208013.4 112229.1 100534.1 315043.5 389293.1 297961.0 0,002 0,002
Adenosylhomocysteinase 114914.1 40278.5 29841.7 70847.1 47868.4 96729.5 0,582 0,173
Actin-7 513312.1 614990.4 272125.6 356455.7 422855.0 436776.3 0,583 0,173
Photosystem II 47 kDa protein 1123112.2 892606.8 1047808.0 1151607.5 1454630.1 1873329.9 0,002 0,002
Photosystem II protein V 38829.1 46227.1 19590.4 52927.9 55935.3 67533.2 0,174 0,069
Photosystem I P700 chlorophyll a apoprotein A2 3493403.0 2583678.5 3431964.2 5972223.7 6671674.3 7296220.5 0,000 0,000
Cytochrome b6 11001.6 3333.6 23939.8 53939.9 36194.8 79898.7 0,001 0,001
ATP synthase CF1 beta subunit 679841.4 1401643.6 1103847.6 472621.9 508287.3 375798.9 0,000 0,000
ATP synthase CF1 alpha subunit 840661.3 1250906.8 372485.8 394510.4 344030.6 256158.5 0,005 0,004

For each quantified protein, the normalized abundances are given in the three individuals of each comparative group, i.e. ‘Tannin’ (T4, T5, and T6) and ‘Control’ (C1, C2, and C3). The Anova p-value and the power q-value are the results of the quantitative analysis performed using ProgenesisQI software.

Table 2c.

Peptidomic list of proteins quantified in the abomasal compartment at 60 minutes of wether digestion of alfalfa hay supplemented in tannins.

Condition Tannin
Condition Control
Description T4 T5 T6 C1 C2 C3 Anova (p) q value
Aquaporin-like transmembrane channel protein 11126.8 22693.2 37081.0 74011.8 90190.1 59915.8
Malate dehydrogenase precursor 36884.3 73079.4 41824.1 14081.2 12192.5 18145.9
Chlorophyll a/b binding protein 3186637.5 3556093.4 4473847.3 7010609.1 6083171.1 14018136.3
Glycolate oxidase, partial 233396.4 450099.4 234807.4 85122.3 153123.6 81345.3
Rubisco activase, partial 483170.2 937792.7 580508.2 443229.8 422350.3 476188.8
Ferredoxin-dependent glutamate synthase, partial 34418.7 64292.8 38866.7 5625.4 12864.9 4004.9
Heat shock protein 70 26200.3 40583.3 26010.2 32437.2 34562.0 49039.4
Pentameric polyubiquitin, partial 267983.8 528200.8 433079.6 322557.6 221439.8 321410.1
Fructose bisphosphate aldolase 208313.6 884946.7 296949.2 192652.2 127326.0 201823.1
Putative glyceraldehyde-3-phosphate dehydrogenase 735020.6 1612765.6 966962.1 409861.4 288234.4 431239.8
Photosystem I P700 chlorophyll a apoprotein A1 439332.4 539277.7 699379.2 1000025.0 1111527.2 1784959.2
Ribulose-1,5-bisphosphate carboxylase/oxygenase 1951203.6 2429332.8 2413332.0 300774.3 283832.0 194295.1
Apocytochrome f 660016.3 977287.3 828789.4 1694342.9 1094491.2 2875504.2
Photosystem II CP43 chlorophyll apoprotein 117568.5 59045.4 124786.3 193296.6 174766.1 341063.8
Histone H3, partial 101891.0 123710.4 47076.9 70381.1 81289.0 38285.8
Ferritin 22450.4 174628.1 42922.7 57585.6 48676.7 26881.5
Photosystem II protein D2 399372.9 491889.0 538907.3 728592.0 671841.7 1615057.1
Ribulose bisphosphate carboxylase small chain, chloroplastic 2930451.6 4343816.2 3979363.0 1095401.7 1332840.8 1137440.0
Photosystem II protein D1 195345.6 200489.9 207922.7 331301.5 465001.1 714769.7
Probable aquaporin TIP-type 178138.0 130487.9 201598.7 340370.4 181964.0 366407.7
Adenosylhomocysteinase 55913.6 115798.9 81187.8 95923.7 42283.9 152799.7
Actin-7 304778.7 729244.7 451907.0 363518.8 266950.7 678686.1
Photosystem II 47 kDa protein 792014.5 888671.8 898258.4 1279347.8 1496798.0 2424064.4
Photosystem II protein V 26384.5 54253.9 38918.1 66718.9 17525.5 108960.3
Photosystem I P700 chlorophyll a apoprotein A2 2951805.5 2550061.7 3906789.3 5676007.1 5584737.3 9240266.1
Cytochrome b6 5720.3 8593.6 20816.5 39401.2 69757.3 50521.5
ATP synthase CF1 beta subunit 808416.9 1256059.1 969204.2 435569.4 608820.1 346295.8
ATP synthase CF1 alpha subunit 463554.6 1129156.3 549360.6 324445.3 295022.4 347029.6

For each quantified protein, the normalized abundances are given in the three individuals of each comparative group, i.e. ‘Tannin’ (T4, T5, and T6) and ‘Control’ (C1, C2, and C3). The Anova p-value and the power q-value are the results of the quantitative analysis performed using ProgenesisQI software.

1.3. Small Intestine Peptidomics

In the small intestine, 63 peptides were identified from the digestion of 11 medicago proteins with at least two unique peptides (Table 3a and via the PRIDE repository with the dataset identifier PXD032973). In this compartment, unique peptides from the two Ribulose proteins (gene name rbcL and RBCS) represent more than 35% of the identified and quantified peptides (23/63 unique peptides). As for the other two compartments, all identification and quantitation results of those peptides are available in the Tables 3b, c for protein data and all the individual raw data acquired during the digestion kinetic in both Control and Tannin groups are available via the PRIDE repository with the dataset identifier PXD032973 (https://www.ebi.ac.uk/pride/archive/projects/PXD032973).

Table 3a.

Peptidomic list of proteins identified in the small intestine compartment during wether digestion of alfalfa hay supplemented in tannins.

Description Mass Accession name Gene name Peptide count Unique peptides Confidence score
Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (chloroplast) 52626.9 1043523999 rbcL 46 19 2095.15
Photosystem I P700 apoprotein A2 (chloroplast) 82427.3 1043524008 psaB 6 3 230.12
Photosystem II CP43 chlorophyll apoprotein (chloroplast) 51907.9 1043524011 psbC 4 4 127.68
Photosystem II protein D2 (chloroplast) 39535.5 1043524012 psbD 5 5 200.89
Cytochrome f (chloroplast) 35300.9 1043524028 petA 5 5 200.49
Photosystem II 47 kDa protein (chloroplast) 55996.1 1043524040 psbB 5 5 181.49
70 kD heatshockprotein, partial 23312.9 1430887 HSP70 2 2 62.00
Ribulose-1,5-bisphosphate carboxylase small subunit, partial 11914.6 16224234 RBCS 4 4 169.92
Tonoplast intrinsic protein homolog MSMCP1 25324.4 2443836 MCP1 4 4 128.37
Chlorophyll a/b binding protein 28312.3 3293555 CARCAB1 9 9 352.00
ATP synthase CF1 beta subunit 52516.0 ANS57890 atpB 3 3 186.3

For each identified protein, the protein name, accession number, gene name, and confidence score were given by MASCOT or Peaks search engines. Mass is the protein molecular weight in Da. Peptide count is the number of peptides used for identification. Unique peptides are the number of peptides belonging to only one protein, and used for quantitation.

Table 3b.

Peptidomic list of proteins quantified in the small intestine compartment at 60 minutes of wether digestion of alfalfa hay supplemented in tannins.

Condition Tannin
Condition control
Description T4 T5 T6 C1 C2 C3 Anova (p) q value
Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (chloroplast) 2687577.2 2818529.4 1226619.2 451227.4 596852.1 379830.1 0,007 0,110
Photosystem I P700 apoprotein A2 (chloroplast) 133604.6 68510.9 88627.7 163463.4 215161.5 184149.7 0,030 0,218
Photosystem II CP43 chlorophyll apoprotein (chloroplast) 2928.9 13558.8 6172.3 3904.2 10309.1 5845.7 0,980 0,788
Photosystem II protein D2 (chloroplast) 33439.6 51182.0 22199.4 38945.7 70052.3 59448.1 0,180 0,496
Cytochrome f (chloroplast) 19619.0 8917.2 18478.9 27646.6 62077.3 55393.5 0,035 0,218
Photosystem II 47 kDa protein (chloroplast) 31756.3 45189.4 26955.8 19559.7 40558.7 29094.3 0,544 0,661
70 kD heatshockprotein, partial 835293.3 1294082.8 477954.3 901645.6 425514.6 861686.5 0,713 0,732
Ribulose-1,5-bisphosphate carboxylase small subunit, partial 39749.9 5976.6 13857.0 10382.3 15178.5 4562.0 0,481 0,661
Tonoplast intrinsic protein homolog MSMCP1 28502.2 30578.8 51972.6 71233.2 45393.7 30251.6 0,457 0,651
Chlorophyll a/b binding protein 349628.0 213941.8 242115.3 185068.8 420252.7 331035.5 0,702 0,732
ATP synthase CF1 beta subunit 45849.6 48924.2 6699.4 1721.9 1317.4 3291.7 0,023 0,218

For each quantified protein, the normalized abundances are given in the three individuals of each comparative group, i.e. ‘Tannin’ (T4, T5, and T6) and ‘Control’ (C1, C2, and C3). The Anova p-value and the power q-value are the results of the quantitative analysis performed using ProgenesisQI software.

Table 3c.

Peptidomic list of proteins quantified in the small intestine compartment at 180 minutes of wether digestion of alfalfa hay supplemented in tannins.

Condition Tannin
Condition control
Description T4 T5 T6 C1 C2 C3 Anova (p value) q value
Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (chloroplast) 146911.2 122501.9 193678.7 91238.0 245590.4 72861.7 0,161 0,729
Photosystem I P700 apoprotein A2 (chloroplast) 50353.7 71072.2 57427.4 53769.6 93352.9 51912.2 0,731 1,000
Photosystem II CP43 chlorophyll apoprotein (chloroplast) 34860.4 16982.5 49548.8 34934.8 4935.1 39881.2 0,552 0,994
Photosystem II protein D2 (chloroplast) 7084.7 17189.1 5698.0 9867.6 38843.0 13022.7 0,288 0,850
Cytochrome f (chloroplast) 7845.0 3759.7 5842.5 5852.9 18523.3 5337.0 0,424 0,983
Photosystem II 47 kDa protein (chloroplast) 5939.2 6256.0 4603.9 2911.6 4532.6 2466.4 0,054 0,729
70 kD heatshockprotein, partial 1949391.3 2740180.3 2339621.9 1902679.8 1195176.9 2751493.7 0,426 0,983
Ribulose-1,5-bisphosphate carboxylase small subunit, partial 1306.9 2669.6 2242.6 758.1 1721.3 1245.2 0,178 0,729
Tonoplast intrinsic protein homolog MSMCP1 8103.3 4525.2 11330.8 10940.8 7761.6 1849.1 0,621 0,994
Chlorophyll a/b binding protein 22083.6 30317.9 19930.5 28820.4 144739.9 99351.7 0,085 0,729
ATP synthase CF1 beta subunit 2423.1 3031.7 5707.4 26.3 786.8 0.0 0,077 0,729

For each quantified protein, the normalized abundances are given in the three individuals of each comparative group, i.e. ‘Tannin’ (T4, T5, and T6) and ‘Control’ (C1, C2, and C3). The Anova p-value and the power q-value are the results of the quantitative analysis performed using ProgenesisQI software.

2. Experimental Design, Materials and Methods

2.1. Experimental Designs

The experiment was conducted at the INRAE Clermont Auvergne Rhône-Alpes center in Theix, France. All animal-related experimental procedures were conducted in accordance with the EU Directive 2010/63/EU, reviewed by the local institutional animal care and use comitee (C2E2A, “Comité d'Ethique pour l'Expérimentation Animale en Auvergne”), and pre-authorized by the French Ministry for Research (approval # 7138-2016092709177605-V5). The protein digestion in the rumen of sheep and in simulated conditions of the abomasum and the small intestine was monitored using a dynamic in vitro digestive system DIDGI® [1] coupled to a digestomic approach (Fig. 1).

Fig. 1.

Fig 1:

Representation of the experimental design, adapted from Sayd et al., 2022. (A) Schematic representation of the ovine digestive tract and (B) the corresponding compartments in the in vitro dynamic digestion system DiDGI®, the abomasum and the small intestine. (C) The sampling and main analysis steps are given for each compartment.

Three rumen-cannulated wethers were fed alfalfa hay and infused daily through the cannula a tannin solution, while three control wethers were infused with water. Standardized ruminal fluid was introduced into a dynamic in vitro digester, which simulated the different digestive compartments in terms of transit rate, pH regulation and digestive enzymes rate [2,3]. Samples were taken along the digestion kinetic and protein degradation in the rumen, the abomasum and the small intestine was determined by the identification and quantitation of peptides after extraction according to Sayd et al. [4].

2.2. LC-MS/MS and Data Analysis

2.2.1. LC-MS/MS Analysis

Peptides (from proteomic (rumen) or digestomic (in vitro digester)) were separated at 400 nl/min (40°C) on a nano HPLC column (Acclaim PepMap RSLC 75um x 25 cm, ThermoScientific) using a gradient of 4-35% acetonitrile (v/v) in 0.1 % (v/v) formic acid within 60 min. The eluted peptides were electrosprayed into nanosources of high resolution mass spectrometers used for this study. Thus, for abomasal and intestinal samples, a LTQ Velos Orbitrap (Thermo scientific) was used and raw data acquired at a resolution of 30000 in full scan (400-2000 m/z). For HCD fragmentation, top 10 method (dynamic exclusion enabled, 60 s) was used and only the most abundant precursors ions in full scan with charge ≥ 2 was activated for fragmentation (collision energy at 37%). For rumen samples, a Q-Exactive HFX was used with 60000 resolution in MS1 (375-1600 m/z) and 15000 in MS/MS (NCE 28) with top 18 method and an exclusion dynamic of 20 s.

2.2.2. Data Analysis

The raw files were processed for quantitation analysis using Progenesis QI software (Nonlinear Dynamics, Waters). For identification, the MS/MS spectra list was exported from the Progenesis QI software as a mascot file (.mgf) to MASCOT (V 2.5) or Peaks (Peaks X+), using the database “Medicago_sativa’’ extracted from NCBI (2020,1099 sequences). The search parameters were set as follow: no enzyme for digestomic analyses and trypsin for proteomic (rumen) analyses, the MS mass tolerance was set at 15 ppm for the peptides and 0.02 Da for the fragments, with a possible mass adduct of methionine oxidation. Peptide identification was validated when ion had a significant Mascot or Peaks score with a false positive rate lower than 0.05. Then, the identification results were re-imported into the Progenesis IQ software for quantitation [5]. Only peptides whose sequence is shared by a single protein were used for the abundance calculation. Quantitative data were normalized based on calculation of scalar factor for each sample which will allow us to recalibrate the sample to a normalization reference run. (normalization method available at: https://www.nonlinear.com/progenesis/qi-for-proteomics/v3.0/faq/how-normalisation-works.aspx. Briefly, this scalar factor, based on all the features intensities required can be represented as αk for each sample: y’i=αkyi, where yi is the measured peptide ion abundance of peptide ion i on sample k, αk is the scalar factor for sample k and y’i is the normalised abundance of peptide ion i on sample k.

Ethics Statements

The experiment was conducted at INRAE Clermont Auvergne Rhône-Alpes centre in France. The experimental procedures on animals were conducted in accordance with the European Union Directive 2010/63/EU, reviewed by the local ethics committee (C2E2A, “Comité d'Ethique pour l'Expérimentation Animale en Auvergne”) and authorised by the French Ministry for Research (no. 7138-2016092709177605-V5).

CRediT Author Statement

Christophe Chambon: Conceptualization, Methodology, Data curation, Writing – original draft preparation, Supervision; Thierry Sayd: Conceptualization, Methodology, Data curation, Writing – original draft preparation; Sylvie Bourillon: Methodology; Laetitia Theron: Methodology, Data curation, Writing – original draft preparation; Vincent Niderkorn: Conceptualization, Methodology, Data curation, Writing – Original draft preparation, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.

Acknowledgments

This work was supported by the H2020 ERA-net project - CORE Organic Cofund - and the cofund from the European Commission, under the project ProYoungStock “Promoting young stock and cow health and welfare by natural feeding systems”. A financial support was also provided by INRAE department ‘Animal physiology and farming’. The authors thank the staff from the Herbipole experimental unit (INRAE Auvergne Rhône-Alpes) for the care of animals.

Data Availability

References

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Data Availability Statement


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