Protein Markers Related to Non-halal Slaughtering Process of Rat as Mammal Animal’s Model Detected Using Mass Spectrometry Proteome Analysis

Alvina Nur Aini(1), Claude Mona Airin(2), Tri Joko Raharjo(3*)

(1) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia
(2) Department of Physiology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Jl. Fauna No. 2 Karangmalang, Yogyakarta 55281, Indonesia
(3) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia; Institute of Halal Industry and System (IHIS), Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
(*) Corresponding Author


Meat produced from non-halal slaughter is forbidden for Moslems. The slaughter methods contribute to the physiological response of animals expressed as different proteome profile. Proteome of two meat obtained from the halal and non-halal slaughter of Wistar rats (Rattus norvegicus) as an animal model was used to search for protein markers related to the slaughter method. The analysis employed Sodium Dodecyl Sulhate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and High-Resolution Mass Spectrometer (HRMS) assisted with Label-Free Quantification (LFQ) Proteome Discoverer software. The non-halal slaughter contributed to the changes in protein expression in animal meat where thirteen proteins were up-regulated and three proteins were specifically identified in the non-halal slaughter, these three proteins are NSFL1 cofactor p47, transketolase, and Von Willebrand. There are three stable peptides identified of those three proteins, SYQDPSNAQFLESIR (m/z = 1755, z = +1) part of NSFL1 cofactor p47, LGQSDPAPLQHQVDVYQK (m/z = 2023, z = +1) part of transketolase, VPLLCTNGSVVHHEVINAMQCR (m/z = 2550, z = +1) part of Von Willebrand. Two of peptides can be targeted as markers in MRM mode LC-MS/MS routine analysis to authentication-halal slaughtering meat. The proposed MRM pair ions are 1755 to 1376, 1262, 1164, and 963, 2033 to 1355, 1016, and 762.


non-halal slaughter; stress response; biomarker; mass spectrometry; label-free proteomics

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[1] Raharjo, T.J., Cahyaningtyas, W., Surajiman, S., Istini, I., and Pranowo, D., 2012, Validation of PCR-RFLP testing method to detect porcine contamination in chicken nugget, Indones. J. Chem., 12 (3), 302–307.

[2] Rohman, A., Erwanto Y., Hossain, M.A.M., Rizou, M., Aldawoud, T.M.S., and Galanakis, C.M., 2021, “The Application of DNAZ-based methods for authentication analysis: Examples in halal and kosher food products” in Food Authentication and Traceability, Eds. Galanakis, C.M., Academic Press, Cambridge, US, 195–213.

[3] Nalazek-Rudnicka, K., Kłosowska-Chomiczewska, I., Wasik, A., and Macierzanka, A., 2019, MRM–MS of marker peptides and their abundance as a tool for authentication of meat species and meat cuts in single-cut meat products, Food Chem., 283, 367–374.

[4] Grandin, T., 2001,” Antemortem handling and welfare” in Meat Science and Applications, Eds. Hui, Y.H., Nip, W.K., Rogers, R.W., and Young, O.A., Marcel Dekker, New York, 221–254.

[5] Bakhsh, A., Ismail, I., Hwang, Y.H., Lee, J.G., and Joo, S.T., 2018, Comparison of blood loss and meat quality characteristics in Korean black goat subjected to head-only electrical stunning or without stunning, Korean J. Food Sci. Anim. Resour., 38 (6), 1286–1293.

[6] Ali, S.A.M., Abdalla, H.O., and Mahgoub, I.M., 2011, Effect of slaughtering method on the keeping quality of broiler chicken meat, Egypt Poult. Sci., 31 (4), 727–736.

[7] Maqsood, S., and Benjakul, S., 2011, Effect of bleeding on lipid oxidation and quality changes of Asian seabass (Lates calcarifer) muscle during iced storage, Food Chem., 124 (2), 459–467.

[8] Mouzo, D., Rodríguez-Vázquez, R., Lorenzo, J.M., Franco, D., Zapata, C., and López-Pedrouso, M., 2020, Proteomic application in predicting food quality relating to animal welfare. A review, Trends Food Sci. Technol., 99, 520–530.

[9] Huang, J.C., Huang, M., Yang, J., Wang, P., Xu, X.L., and Zhou, G.H., 2014, The effects of electrical stunning methods on broiler meat quality: Effect on stress, glycolysis, water distribution, and myofibrillar ultra-structures, Poult. Sci., 93 (8), 2087–2095.

[10] Addeen, A., Benjakul, S., Wattanachant, S., and Maqsood, S., 2014, Effect of Islamic slaughtering on chemical compositions and post-mortem quality changes of broiler chicken meat, Int. Food Res. J., 21 (3), 897–907.

[11] Nielsen, S.S., Alvarez, J., Bicout, D.J., Calistri, P., Depner, K., Drewe J.A., Garin-Bastuji, B., Rojas, J.L.G., Schmidt, C.G., Herskin, M., Michel, V., Chueca, M.A.M., Roberts, H.C., Sihvonen, C.G., Spoolder, H., Stahl, K., Velarde, A., Viltrop, A., Candani, D., Van der Stede, Y., and Winckler, C., 2020, Welfare of cattle during killing for purposes other than slaughter, EFSA J., 18 (11), e06312.

[12] Żurek, J., Rudy, M., Kachel, M. and Rudy, S., 2021, Conventional versus ritual slaughter–ethical aspects and meat quality, Processes, 9 (8), 1381.

[13] Velarde, A., Gispert, M., Diestre, A., and Manteca, X., 2013, Effect of electrical stunning on meat and carcass quality in lambs, Meat Sci., 63 (1), 35–38.

[14] Yardimci, M., 2019, Comparison of the stunning and non-stunning slaughtering methods in the light of the current knowledge, J. Vet. Anim. Res., 2 (3), 1–5.

[15] Samah, N.A., Amid, A., and Yusof, F., 2011, Overexpression of voltage-dependent anion channel 2 (VDAC2) in muscles of electrically stunned chickens, IIUM Eng. J., 12 (4), 213–222.

[16] Amid, A., Samah, N.A., and Yusof, F., 2012, Identification of troponin I and actin, alpha cardiac muscle 1 as potential biomarkers for hearts of electrically stimulated chickens, Proteome Sci., 10 (1), 1.

[17] Kiran, M., Naveena, B.M., Smrutirekha, M., Baswa Reddy, P., Banerjee, R., Praveen, K.Y., Venkatesh, C., and Rapole, S., 2019, Traditional halal slaughter without stunning versus slaughter with electrical stunning of sheep (Ovis aries), Meat Sci., 148, 127–136.

[18] Salwani, M.S., Adeyemi, K.D., Sarah, S.A., Vejayan, J., Zulkifli, I., and Sazili, A.Q., 2015, Skeletal muscle proteome and meat quality of broiler chickens subjected to gas stunning prior slaughter or slaughtered without stunning, CyTA-J. Food, 14 (3), 375–381.

[19] Yates, J.R., 2013, The revolution and evolution of shotgun proteomics for large-scale proteome analysis, J. Am. Chem. Soc., 135 (5), 1629–1640.

[20] Aini, A.N., 2019, Identifikasi Protein Penanda Spesifik Metode Penyembelihan Hewan Menggunakan Spektrometri Massa Resolusi Tinggi, Thesis, Universitas Gadjah Mada, Indonesia.

[21] Szklarczyk, D., Gable A.L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., Simonovic, M., Doncheva, N.T., Morris, J.H., Bork, P., Jensen, L.J., and von Mering, C., 2019, STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets, Nucleic Acids Res., 47 (D1), D607–613.

[22] Orduna, A.R., Husby, E., Yang, C.T., Ghosh, D., and Beaudry, F., 2017, Detection of meat species adulteration using high-resolution mass spectrometry and a proteogenomics strategy, Food Addit. Contam., Part A, 34 (7), 1110–1120.

[23] Kanehisa, M., and Goto, S., 2000, KEGG: Kyoto encyclopedia of genes and genomes, Nucleic Acids Res., 28 (1), 27–30.

[24] Doherty, M.K., McLean, L., Hayter, J.R., Pratt, J.M., Duncan, H.L., Robertson, D.H., El-Shafei, A., Gaskell, S.J., and Beynon, R.J., 2004, The proteome of chicken skeletal muscle: Changes in soluble protein expression during growth in a layer strain, Proteomics, 4 (7), 2082–2093.

[25] The UniProt Consortium, 2019, UniProt: A worldwide hub of protein knowledge, Nucleic Acid Res., 47 (D1), D506–515.

[26] Feng, Y.H., Zhang, S.S., Sun, B.Z., Xie, P., Wen, K.X., and Xu, C.C., 2020, Changes in physical meat traits, protein solubility, and the microstructure of different beef muscles during post-mortem aging, Foods, 9 (6), 806.

[27] Astuti, P., Putro, C.P.C., Airin, C.M., Sjahfirdi, L., Widiyanto, S., and Maheshwari, H., 2014, Comparison of process slaughtered on beef cattle based on level of cortisol and Fourier Transform Infrared Spectroscopy (FTIR), Int. J. Anim. Vet. Sci., 8 (12), 876–879.

[28] Adeva-Andany, M.M., González-Lucán, M., Donapetry-García, C., Fernández-Fernández, C., and Ameneiros-Rodríguez, E., 2016, Glycogen metabolism in humans, BBA Clin., 5, 85–100.

[29] Bader, S., Meyer-Kühling, B., Günther, R., Breithaupt, A., Rautenschlein, S., and Gruber, A.D., 2014, Anatomical and histological pathology induced by cervical dislocation following blunt head trauma for on-farm euthanasia of poultry, J. Appl. Poult. Res., 23 (3), 546-556.

[30] Nakyinsige, K., Fatimah, A., Aghwan, Z.A., Zulkifli, I., Goh, Y.M., and Sazili, A.Q., 2014, Bleeding efficiency and meat oxidative stability and microbiological quality of New Zealand white rabbits subjected to halal slaughter without stunning and gas stun killing, Asian-Australas. J. Anim. Sci., 27 (3), 406–413.

[31] Abdela, N., Jilo, K., Siraj, S., Adem, J., and Mohammed, A., 2016, Impact of stress on health and productivity of animal: A review, J. Nat. Sci. Res., 6 (9), 45–51.

[32] Krebs, J., Agellon, L.B., and Michalak, M., 2015, Ca(2+) homeostasis and endoplasmic reticulum (ER) stress: An integrated view of calcium signaling, Biochem. Biophys. Res. Commun., 460 (1), 114–121.

[33] Zeng, J., Shu, Z., Liang, Q., Zhang, J., Wu, W., Wang, X., and Zhou, A., 2022, Structural basis of von Willebrand factor multimerization and tubular storage, Blood, 139 (22), 3314–3324.

[34] Rauh, M., 2012, LC-MS/MS for protein and peptide quantification in clinical chemistry, J. Chromatogr. B, 883-884, 58–67.

[35] You, J., Willcox, M.D., Madigan, M.C., Wasinger, V., Schiller, B., Walsh, B.J., Graham, P.H., Kearsley, J.H., and Li, Y., 2013, Tear fluid protein biomarkers, Adv. Clin. Chem., 62, 151–196.


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