Arc. Tierz., Dummerstorf 51 (8) Special Issue, 62-68 1 Animal Breeding and Genomics Centre (ABGC), Wageningen University and Researc Centre Animal Sciences Group (ASG-WUR), Te Neterlands 2 Hypor BV, A Hendrix Genetics Company, Best, Te Neterlands 3 ABGC, ASG-WUR, Wageningen, Te Neterlands 4 Clinical Sciences of Companion Animals, Faculty of Veterinary Science, Utrect University, Te Neterlands MARINUS F. W. TE PAS 1, JAAP JANSEN 1, KONRAD C. J. A. BROEKMAN 2, HENNY REIMERT 1 and HENRI C. M. HEUVEN 3,4 Post mortem proteome degradation profiles of longissimus muscle in Yorksire and Duroc (Post mortem Proteom-Degradationsprofile vom M. longissimus dorsi bei den Rassen Yorksire und Duroc) Abstract Post mortem enzymatic proteolysis canges toug intact muscle tissue into tender meat. Te extend of muscle protein proteolysis greatly determines te quality of te meat: inadequate proteolysis produces toug meat, too muc proteolysis is associated wit increased drip loss wic also reduces tenderness and juiciness of te meat. Wile information on te proteolysis of several specific proteins is available, knowledge regarding te progress of proteolysis on te entire proteome of muscle tissue is limited. Furtermore, breed-specific differences ave received little attention. Terefore, tis study we investigated breakdown profiles of te longissimus proteome of Yorksire and Duroc pigs. Multiple samples of longissimus muscle tissue of five pigs of eac breed were collected and drip loss, cooking loss, sear force, as well as proteome profiles, using SELDI-TOF tecnology, were determined after 1, 2, 3, 7, and 1 days. Drip loss increased wit ageing and was sligtly iger in Yorksire tan in Duroc longissimus wile cooking loss was uncanged during ageing and similar in bot breeds. Sear force decreased wit ageing time. Sear force decreased more in Yorksire compared to Duroc, but profiles differed between animals. Te number of peptides detected wit SELDI-TOF was iger in York tan in Duroc. It suggests a relationsip between ageing-related levels of proteolysis and meat quality traits. Keywords: proteomics, meat, proteolysis Zusammenfassung Enzymatisce post mortem Proteolyse überfürt intaktes Muskelgewebe in Fleisc. Dabei fürt unzulänglice Proteolyse zu zäem Fleisc, zu starke Proteolyse fürt zu erötem Tropfsaftverlust, der auc die Weiceit und Saftigkeit des Fleisces verringert. Wärend Informationen über die Proteolyse einiger spezifiscer Proteine voranden sind, ist das Wissen über Veränderungen des Proteoms wärend der Proteolyse begrenzt. Außerdem sind rassenspezifisce Untersciede biser wenig berücksictigt worden. Diese Studie zielt darauf ab, die Profile der M. longissimus Proteome wärend der Proteindegradation bei den Rassen Yorksire und Duroc zu analysieren. Gewebeproben des M. longissimus von je fünf Scweinen der beiden Rassen wurden 1, 2, 3, 7 und 1 Tage nac der Sclactung gewonnen zur Bestimmung von Tropfsaftverlust, Kocverlust und Scerkraft sowie der Darstellung von Proteomprofilen mittels SELDI-TOF. Der Tropfsaftverlust eröte sic mit dem Altern und war beim Yorksire etwas öer als beim Duroc. Beim Kocen blieben die Verluste unverändert wärend des Alterns und änlic in beiden Rassen. Die Scerkraft verringerte sic mit der Zeit. Die Abname war beim Yorksire öer. Die Zal der Peptiden, die mit SELDI-TOF ermittelt wurden, war beim Yorksire öer als beim Duroc. Dies deutet auf einen Zusammenang zwiscen alterungsabängigen Niveaus der Proteolyse und Fleiscqualitätsmerkmalen in. Sclüsselwörter: Proteomics, Fleisc, Proteolyse Introduction Skeletal muscle tissue formation in mammals is a prenatal process (REHFELDT et al., 4; STICKLAND et al., 4). After birt te skeletal muscle develops furter by lengt and ypertropic growt (TE PAS and SOUMILLION, 1). During life of te animal te muscle determines mobility and stability of te animal. Muscle tissue consists of many cell types including muscle fibers, adipocytes, neurons, endotelial
63 cells, etc. Muscle mass is determined mainly by muscle fibers. Due to different functional requirements muscles differ in muscle fiber type composition. Muscle fiber types differ in metabolic activity and can be recognized by te expression of muscle fiber type-specific proteins (REGGIANI and MASCARELLO, 4). After slaugtering muscle tissue becomes meat. Meat as different caracteristics tan muscle tissue during life. Post mortem processes called ageing of meat cange te alive-muscle caracteristics into eatable meat caracteristics. A major factor in ageing is te proteolysis of proteins tat were required for functioning during life but wic induce tougness in meat (KOOHMARAIE 1996; HOPKINS et al., 2). Due to muscle fiber type differences te proteolysis may differ between muscles. Due to selection proteolysis may also differ between breeds even witin a single muscle. Relatively little knowledge of te canges in te total tissue proteome during ageing (LAMETSCH, 1, 2, 3), and muscle-specific and breed-specific differences ave been reported. Terefore, we initiated tis study by analyzing te proteome breakdown profiles during ageing in longissimus muscle in two pig breeds differing in pork quality parameters. Materials and Metods Animals and sampling Five Yorksire and five Duroc pigs, raised under similar conditions, were slaugtered at te same day. Growt rate data and backfat data were available for all animals. On te day of slaugter (day ) longissimus and am muscle tissue samples were taken from te rigt carcass side after te carcasses were rapid cilled for 3 min. Samples were taken 1.5 after slaugter. Te longissimus muscle was sampled between vertebrae tree and four. Part of te samples were weiged and stored in an open box at 4 for drip loss measurements. Starting weigts were about -3 g eac. Part of te samples was snap frozen in liquid nitrogen and stored at -8 for future proteomics measurements. All oter samples were taken at day 1 from te left carcass alf wic was stored intact at 4 New samples for drip loss measurements were taken and treated exactly as described above. Oter samples were taken from between vertebrae four-five, etc. until vertebrae nine-ten. Tese samples were stored in a plastic bag at 4 and used for determination of cooking loss and sear force as described below. Meat quality traits In te slaugterouse ph at 24, conductivity and meat color (Japanese color scale and Minolta) were determined at te day after slaugter. Drip loss was determined by weiging te samples on te day of taking te samples, and reweiging tem after drying wit a tissue on days 1, 2, 3, 4, 7 and 1. Cooking loss was determined by weiging longissimus samples, eating at 7 in a sealed plastic bag for 1 and cooling under running tap water for 1 at te same days. After weiging, te cooked samples were stored in a sealed plastic bag at - until sear force determination (HONIKEL, 1998). Eac sample was measured six times by taking six sub-samples. Proteomics profiles Samples for proteomics were removed from te freezer and te water-soluble fraction of proteins was isolated. Samples were weiged (3-5 mg), placed in 1.5 ml lysis
64 buffer (1 mm Tris-HCl ph 7.25, 1 mm KCl, 2 % [v/v] Triton X-1, 1 mm PMSF), omogenized on ice using an Ultra-Turrax T25 wit dispersion tool S25N-1G (9, rpm, s) and incubated on ice for 1 and vigorously saken occasionally. Subsequently, te omogenate was cleared by centrifugation (Eppendorf centrifuge, 3 min, 15, rpm, 4 max d), te resulting supernatant was transferred to a new tube and stored at -8 until furter processing. Te protein concentration of te cleared supernatant was determined using te Bio-Rad DC assay (Bio-Rad, Veenendaal, Te Neterlands). SELDI-TOF (Biorad) analysis was performed on te Protein Cip System Series 4 equipment and te data were analyzed using Cipergen Express Software release 3..6. After an initial screening te IMAC3-Cu (IMAC3 EDM buffer kit), CM1 (ph5 buffer) and Q1 (ph 6 buffer) cip array types and binding buffer combinations were cosen. Te different ProteinCip arrays were equilibrated wit te respective binding buffers containing.1 % Triton. Equal amounts of cleared lysate sample protein was loaded on te arrays togeter wit te respective binding buffers (total volume 25 μl) using a Bioprocessor (Biorad) and incubated for one our at room temperature wit continuous saking. After removal of te diluted sample extracts, te arrays were subsequently wased wit te respective binding buffers followed by a quick rinse wit deionised water. After air-drying te arrays at room temperature energy absorbing matrix sinapinic acid (SPA) in 5 % acetonitril and.5 % trifluoroacetic acid was added to eac spot. Mass analysis of te bound proteins was performed using Protein Cip System Series 4 (Biorad) in positive operating mode. Mass spectra were collected by te accumulation of lasersots at a laser intensity of 3 nj. Te igest mass to acquire was set at 15, kda; Focus Mass setting was 5, kda. Te instrument was calibrated by using te All-in-one Protein Standard II (Biorad). Te EDM tool of te software was used to create clusters using te settings: S/n-ratio 3 and %-age occurrence of peak features= %. Tis means tat clusters were created wen 1 of 5 of te spectra contained a peak wit an s/n ratio of 3. Results Meat quality traits Drip loss did not differ between te Yorksire and Duroc breeds in te longissimus muscle, but was significantly different between days of sampling: day of slaugter and day 1 (different carcass sides) as well as over time. After correction for correlated residuals Yorksire carcasses ad.15 % more drip loss tan Duroc carcasses (not significant). Drip loss increases wit day (Figure 1). Drip loss (%) 8 6 4 2 Mean drip loss 2 4 6 8 1 Yorksire, LD Duroc, LD Yorksire, am Duroc, am Fig. 1: Mean drip loss of longissimus and am muscles of five Duroc and five Yorksire carcasses from slaugter to ten.
65 Cooking loss sowed similar results as Yorksire and Duroc breeds did not differ, but bot breeds sowed a significant day effect. Yorksire pigs sowed on average.364 % more cooking loss tan Duroc pigs (not significant), but tis difference increased over time (Figure 2). Cooking loss (%) 38 36 34 32 3 28 26 24 22 Mean cooking loss 2 4 6 8 1 12 Yorksire, LD Duroc, LD Yorksire, am Duroc, am Fig. 2: Mean cooking loss of longissimus and am muscles of five Duroc and five Yorksire carcasses from slaugter to ten Sear force repeated measurements on a single sample were not significantly different indicating good repeatability of te measurements. Sear force was also not different between Yorksire and Duroc breeds wit Yorksire pigs being a little less tender tan Duroc. Sear force decreased wit day in a non-linear pattern (Figure 3). LD Yorksire am Yorksire 7 6 sear 5force sear force 4 3 1 1 2 3 7 1 days post post mortem mortem 952ld 953ld 954ld 956ld 963ld sear force 6 5 4 3 1 1 2 3 7 1 952 953 954 956 963 LD Duroc am Duroc sear force 9 8 7 6 5 4 3 1 1 2 3 7 1 958 ld 959 ld 96 ld 961 ld 964 ld days post post mortem mortem Fig. 3: Sear force measurements (averaged over 6 sub-samples) of longissimus and am muscles of five Duroc and five Yorksire carcasses from slaugter to ten. sear force 7 6 5 4 3 1 1 2 3 7 1 Proteomics profiles Peak eigt was taken as a measure for expression level. Te results sow a profile of peaks representing different proteins. On average te profiles of Yorksire pigs sowed more peaks tan te profiles of Duroc pigs (data not sown). 958 959 96 961 964
66 Analyzing te results in time suggest four different profiles of protein expressions (Figure 4). In figure 4 representative examples of protein features representative for te four classes of profiles are given. (1) Profile one suggests tat te expression level at day zero is eiter very low or not existing. After day one te expression increases in time and continues to increase until te end of te experiment on day 1. Te increase may eiter start on day one or on a later day indicating variation witin tis profile between individual peaks. Tis profile suggests tat te peak is a degradation product. (2) Profile two start similar to profile one but reaces a maximum after wic te expression decreases. Tis profile suggests tat te peak is a degradation product tat is degraded also. (3) Profile tree is te opposite of profile one: starting a ig expression te expression decreases. Te profile suggests tat te peak is an original muscle protein tat is degraded. (4) Profile four indicates tat te expression of te protein is uncanged over time suggesting tat te protein is an original muscle protein tat is not degraded. 14 1 1 8 6 4 Profile type 1 2 4 6 8 1 1 1 8 6 4 Profile type 2 2 4 6 8 1 6 5 4 3 1 Profile type 3 4 35 3 25 15 1 5 Profile type 4 2 4 6 8 1 2 4 6 8 1 Fig. 4: Longissimus muscle proteome degradation profile types. Type 1: Expression at slaugter is low and increases during degradation. Te time of te start of te increased expression varies between protein fragments (size). Suggestive for a degradation product. Type 2: Expression at slaugter is low and increases during degradation, but followed by a concomitant decrease. Te times of te start of te increased expression and te decrease varies between fragments. Suggestive for a degradation product tat itself is degraded furter. Type 3: Expression at slaugter is ig and decreases during degradation. Te time of te start of te decreased expression varies between fragments. Suggestive for a muscle protein tat is degraded. Type 4: Peak wit a stable expression. Suggestive for a muscle protein tat is not degraded during meat aging. Discussion Conversion of muscle to meat is regulated by complex interactions of biocemical processes tat take place during postmortem storage of te carcass (OUALI, 1992; KOOHMARAIE, 1996). Post mortem proteolysis is one suc important process.
67 Especially te degradation of titin and nebulin, bot located in te I-band, is of great interest as electron microscopy of post-mortem meat samples as sown tat te major fragmentation of te myofibrils occurs in te I-band area (TAYLOR et al., 1995; BOYER-BERRI et al., 1998; HUFF-LONERGAN, 1995). Several studies ave sown troponin T to be degraded postmortem, and tis degradation is believed to be closely related to meat tenderness (OUALI, 1992; HO et al., 1994). However, te degradation of te entire proteome is poorly known. Present proteomics tecniques enable te study of te proteome. Our results sow tat proteolysis continues during te wole period of sampling, i.e. until ten days after slaugtering te animal. Wile meat quality data sowed tat differences between animals and breeds are minimal, animals and breeds differed in proteome degradation profiles of te longissimus muscle proteome. Tis suggests tat differences in meat quality between carcasses are related to differences in te degradation of te proteome. Differential rate of degradation or differences in te order of degradation of proteins may affect eating quality traits since pork is mostly partly degraded at te moment of consumption. Most fres pork is consumed witin a week. Caracterization of te peptides leading to identification of te proteolytically cleaved muscle proteins may yield more insigt in te process of tenderization. Furter detailed studies are required wit respect to tis subject. From our study it can be concluded tat SELDI-TOF is a igly efficient metod for te detection of muscle protein degradation products wic are generated during te meat tenderization process. Correlation of meat proteome degradation profiles wit oter meat quality data may lead to te identification of Biomarkers for meat quality traits tat can be used in te slaugterouse. Development of Biomarkers tat can be used during breeding or fattening livestock is also envisioned. Acknowledgements Tis work was supported by a SIM-grant 4434.6291. from te Director of te Animal Production Division. References BOYER-BERRI, C.; GREASER, M.L.: Effect of postmortem storage on te Z-line region of titin in bovine muscle. J. Anim. Sci. 76 (1998), 134-144 HO, C.Y.; STROMER, M.H.; ROBSON, R.M.: Identification of te 3 kda polypeptide in post mortem skeletal muscle as a degradation product of troponin-t. Biocemie 76 (1994), 369-375 HONIKEL, K.O.: Reference metods for te assessment of pysical caracteristics of meat. Meat Sci. 49 (1998), 447-457 HOPKINS, D.L.; THOMPSON, J.M.: Factors contributing to proteolysis and disruption of myofibrillar proteins and te impact on tenderisation in beef and seep meat. Aust. J. Agric. Res. 53 (2), 149-166 HUFF-LONERGAN, E.; PARRISH, F.C.; ROBSON, R.M.: Effect of post mortem aging time, animal age, and sex on degradation of titin and nebulin in bovine longissimus muscle. J. Anim. Sci. 73 (1995), 164-173 KOOHMARAIE, M.: Biocemical factors regulating te tougening and tenderization processes of meat. Meat Sci. 43 (1996), 193-1 LAMETSCH, R.; BENDIXEN, E.: Proteome analysis applied to meat science: caracterizing postmortem canges in porcine muscle. J. Agic. Food Cem. 49 (1), 4531-4537
68 LAMETSCH, R.; ROEPSTORFF, P.; BENDIXEN, E.: Identification of Protein Degradation during Post-mortem Storage of Pig Meat. J. Agric. Food Cem. 5 (2), 558-5512 LAMETSCH, R.; KARLSSON, A.; ROSENVOLD, K.; ANDERSEN, H.J. ROEPSTORFF, P.; BENDIXEN, E.: Postmortem Proteome Canges of Porcine Muscle Related to Tenderness. J. Agric. Food Cem. 51 (3), 6992-6997 OUALI, A.: Proteolytic and pysicocemical mecanisms involved in meat texture development. Biocimie 74 (1992), 251-265 REGGIANI, C.; MASCARELLO, F.: Fibre type identification and functional caracterization in adult livestock animals. In: Muscle Development of Livestock Animals Pysiology, Genetics, and Meat Quality (Editors, M.F.W. te Pas, M.E. Everts, and H.P. Haagsman). CABI publisers, Wallingford, Oxfordsire, UK, 4, 39-68 REHFELDT, C.; FIEDLER, I.; STICKLAND, N.C.: Number and size of muscle fibres in relation to meat production. In: Muscle Development of Livestock Animals Pysiology, Genetics, and Meat Quality (Editors, M.F.W. te Pas, M.E. Everts, and H.P. Haagsman). CABI publisers, Wallingford, Oxfordsire, UK, 4, 1-38 STICKLAND, N.C.; BAYOL, S.; ASHTON, C.; REHFELDT, C.: Manipulation of muscle fibre number during prenatal development. In: Muscle Development of Livestock Animals - Pysiology, Genetics, and Meat Quality (Editors, M.F.W. te Pas, M.E. Everts, and H.P. Haagsman). CABI publisers, Wallingford, Oxfordsire, UK, 4, 69-82 TAYLOR, R.G.; GEESINK, G.H.; THOMPSON, V.F.; KOOHMARAIE, M.; GOLL, D.E.: Is Z-disk degradation responsible for postmortem tenderization? J. Anim. Sci. 73 (1995), 1351-1367 TE PAS, M.F.W.; SOUMILLION, A.: Te use of pysiologic and functional genomic information of te regulation of te determination of skeletal muscle mass in livestock breeding strategies to enance meat production. Current Genomics 2 (1), 285-34 Corresponding autor: MARINUS F.W. TE PAS Animal Breeding and Genomics Centre (ABGC) Animal Sciences Group, Wageningen University and Researc Centre (ASG-WUR) P.O. Box 65 8 AB Lelystad Te Neterlands email: marinus.tepas@wur.nl