Food Chemistry 150 (2014) 137–144
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Analytical Methods
LC MS/MS identification of large structural proteins from bull muscle and their degradation products during post mortem storage Guojie Wu a,⇑, Stefan Clerens b, Mustafa M. Farouk a a b
AgResearch, Ruakura Research Centre, Hamilton, New Zealand AgResearch, Lincoln Research Centre, Christchurch, New Zealand
a r t i c l e
i n f o
Article history: Received 21 January 2013 Received in revised form 17 October 2013 Accepted 26 October 2013 Available online 2 November 2013 Keywords: Protein identification Muscle structural proteins Myosin super-family Post mortem storage SDS–PAGE LC–MS/MS
a b s t r a c t Large proteins (>100 kDa) in bovine M. longissimus dorsi and their degradation products during post mortem ageing were investigated by gel electrophoresis and LC–MS/MS analysis. Seventeen protein bands from SDS–PAGE were analysed and 26 proteins were identified. Intact titin, nebulin and filamin were shown to break down during post mortem ageing of meat. A number of myosin super-family members were revealed on SDS–PAGE. Myosin heavy chain 1 (MYH1), myosin heavy chain 2 (MYH2), and myosin heavy chain 7 (MYH7) were distributed broadly across the bands in the forms of cross-linked/aggregated polymers, and also as fragments. Three myomesin family members: myomesin 1 (185 kDa isoform 1), myomesin (M-protein) 2, 165 kDa, and myomesin family member 3, were identified in the muscle samples. Several other proteins such as synemin, myosin binding protein C (C-protein), glycogen debranching enzyme and ryanodine receptor 2 were also identified. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Meat tenderness has been well documented in relation to changes in muscle structural proteins during post mortem storage (Anderson & Parrish, 1989; Huff Lonergan, Zhang, & Lonergan, 2010; Lametsch et al., 2003; Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). Small structural proteins (100 kDa) meat proteins have been studied by immunochemistry and other techniques (Huff-Lonergan, Mitsuhashi, Beekman, et al., 1996; Shibata et al., 2009; Taylor et al., 1995). This fact could be due to the lack of knowledge on their structure and functions or to the low abundance and poor separation of these proteins, leading to difficulties in their characterization. One-dimensional gel electrophoresis (1-DE) is widely used for analysing muscle protein changes during post mortem storage (Di Luca, Mullen, Elia, Davey, & Hamill, 2011; Huang, Huang, Xu, & Zhou, 2009; Lomiwes et al., 2013; Watanabe & Devine, 1996; Zapata, Zerby, & Wick, 2009). We previously (Farouk, Mustafa, Wu, & Krsinic, 2012) identified titin, nebulin and filamin from beef M. semimembranosus separated by 1D SDS–PAGE. Recently we have shown meat tenderness to be closely linked to ultimate meat pHu by variable rate of degradation of high and low molecular weight myofibrillar proteins during post mortem ageing of bull beef M. longissimus dorsi (LD) muscle (Lomiwes, Farouk, Wu, & Young, 2014; Lomiwes et al., 2013). In the present work, we used liquid
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chromatography coupled to tandem mass spectrometry (LC–MS/ MS) to identify all of the high molecular weight proteins and their degradation products during post mortem storage, in order to determine the changes of proteins in muscle during post mortem aging and its relationship with meat quality.
2. Materials and methods 2.1. Tissue samples Bulls (n = 18, Angus or Angus-cross, approximately 250–300 kg wet carcass weight and 18 to 24 months of age) were slaughtered at a commercial export meat processing facility in accordance with New Zealand and relevant overseas market access regulatory requirements. The bulls were stunned using head-only reversible electrical stunning. Muscle samples were removed from the anterior region (12th to 13th rib region) of M. longissimus dorsi (LD) of each animal. One small portion of each LD was taken and immediately frozen in liquid nitrogen to serve as the zero time point. The remaining muscles were allowed to enter rigor at 15 °C and assigned randomly to different aging periods (2, 7, 21 and 28 days). These samples were vacuum-packed and stored at 1.5 °C. At each time point, samples were transferred into a freezer ( 30 °C) until analysis.
2.2. Extraction of muscle/meat proteins Seven samples were taken from each animal. Total muscle proteins were extracted from 126 differently aged muscle/meat samples. The extraction procedure was used as described in our recent study (Lomiwes, Farouk, Wu, et al., 2014). Protein contents of the extracts were measured using the Bio-Rad RC-DC Protein Assay Kit according to the manufacturer’s instructions (Bulletin #4110107 Rev A, Bio-Rad).
2.5. LC–MS/MS analysis Seventeen protein bands separated by SDS–PAGE was identified by LC–MS/MS as described in previously (Farouk et al., 2012) with the following additional details: Eighty microliter of 0.2 lM sequencing grade porcine trypsin (Promega, USA) in 50 mM ammonium hydrogen carbonate (NH4HCO3) with 10% acetonitrile, was added to the gel pieces, and followed by brief vortexing and overnight incubation at 37 °C. Both the C18 precolumn and the analytical column were from Varian Microsorb (United States). MS/MS was programmed to acquire the three most intense peaks, restricted to 2+, 3+ and 4+ compounds in the m/z 400–1200 range. The mass spectrometer was used in positive ion, nano-electrospray model. The spray voltage was 2200 V and the collision gas was nitrogen, set at 4 (arbitrary units). MS/MS was acquired from m/z 50–2000. Enzyme semitrypsin was specified to allow identification of peptides that had undergone non-specific backbone cleavage, either during post mortem storage or during sample preparation. The database was NCBInr. The criteria of validation of search results were similar to those used by Zapata et al. (2009) with slight modification. In brief, all data identified as originating from trypsin and keratin, and those based on only one or two unique sequences, were removed, thereby retaining only identifications corresponding to bovine sequences with a minimum two unique peptides in the accepted data set. 3. Results and discussion Fig. 1 shows a representative map of the large proteins with molecular weight over 100 kDa separated by SDS–PAGE. Seventeen bands were present in the gel. The identification results are shown in Table 1. Gel band numbers were assigned on the basis of decreasing molecular weight. 3.1. Protein bands with molecular weight above 600 kDa: Bands 1–6
2.3. SDS–PAGE Sample preparation was as previously reported (Lomiwes, Farouk, Wiklund, & Young, 2014). In the present study, 5% Tris–HCl separating gels (Criterion precast gels, Bio-Rad) were used to separate the larger proteins (>100 kDa). Each sample was loaded at 100 lg protein per well to give high intensity bands. Electrophoresis was conducted at a constant current of 5 mA/gel for 16.5 h. The running buffer, consisting of 25 mM Tris, 192 mM glycine and 0.1% SDS, was used in both the 5% gels and the upper and lower buffer chambers of the Criterion Cell system (Bio-Rad). Bio-Rad prestained kaleidoscope molecular weight standards and HiMark™ pre-stained higher molecular weight protein standards (Invitrogen, USA) were employed (10 ll per well) to aid in identification of protein bands in the gels. Gels were Coomassie stained (17% ammonium sulphate, 2% phosphoric acid, 34% methanol and 0.04% Colloidal Coomassie G-250) for 48 h at room temperature.
2.4. Quantity analysis of protein bands The gels were scanned with a GS700 Calibrated Densitometer Scanner (Bio-Rad) and quantity was analysed using Quantity One software (4.6.5) from Bio-Rad. The optical density (OD) of each band is compared relative to the sample at 0 day post mortem except the band 2 and 12 which are relative to the samples at 2 days and 7 days post mortem aging, respectively, as they were generated at that time points. The results were shown as relative quantity (%).
Bands 1, 2 and 3 (present mainly in lanes of sample at 0 and after 2 days post mortem) were identified successfully as titin (gi|29471578), with hundreds (314–559) of unique peptides assigned to this molecule (Fig. 1 and Table 1) each representing a single protein. Titin in band 1 represents intact titin (T1), which decreased in intensity with post mortem ageing (Figs. 1 and 2). This result was consistent with previous publications (Ho et al., 1996; Huff-Lonergan, Mitsuhashi, Parrish, et al., 1996; Watanabe & Devine, 1996). Band 2 was more abundant in high pHu than in intermediate and low pHu muscles (unpublished data). This band increased in intensity in aged meat and became more intense and covered a wider range of molecular weights (band 3) at 28 days post mortem (Figs. 1 and 2), indicating the formation of degradation products. Bands 2 and 3 consist largely of titin degradation products (T2, 2400 kDa) (Ho et al., 1996; Huff-Lonergan, Mitsuhashi, Parrish, et al., 1996). As shown in Table 1, band 4 was identified as a mixture of six proteins. Titin is ranked in the top. The assignment of one component of this band as a titin fragment comes from previous identifications of this band as a 1200 kDa degradation product of intact titin (Huff-Lonergan, Mitsuhashi, Parrish, et al., 1996; Takahashi, 1996). Ho et al. (1996) demonstrated that this 1200 kDa band was present soon after death (0 and 1 day post mortem) and decreased with ageing time. Our gels showed similar results. This protein was identified as a different database entry (gi|297465038) than the top three titin bands (accession number gi|297471578). However both database entries are the same size and the identified peptides are distributed across the complete
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Fig. 1. Representative lanes from SDS–PAGE gel of large proteins from beef muscle during post mortem storage. All numbered bands were submitted for LC–MS/MS analysis. B = the abbreviation of band; MHC = myosin heavy chain. B1, intact titin; B2, T2; B3, T2; B4,titin isoform/MYH1/MYHIIa/MYH7/myosin-3 like; B5, Intact nebulin/titin/MYH1/ MYHIIa/MYH7/myosin-3 like; B6, MYH2/MYH1; B7, MYH7/MYH2/nebulin; B8, MYH1/MYH2/titin/MYH7/Myh6/nebulin/myosin-3 like/epiplakin 1; B9, nebulin/titin/MYH2/ MYH1/MYH7/Myh6/ryanodine receptor 2-like/cytoplasmic dynein 1 heavy chain 1/b-lactoglobulin; B10, MYH1/MYH7/nebulin; B11, Intact filamin; B12, c-isoform 3/MYH1; B13, Myomesin 1/myomesin family member 3/MYH2/MYH1/myosin-8/MYH7/Myh6/synemin/myomesin 2; B14, Myomesin 2/MYH1/MYHIIa/MYH7/myomesin 1/glycogen debranching enzyme; B15, MYH2/MYH1/myosin-8/myosin binding protein C/glycogen debranching enzyme/Myh 6/MYH7/myomesin 2; B16, Myosin binding protein C/MYH1/MYH2/MYH7/ glycogen debranching enzyme; B17, MYH2/MYH1/MYH7/collagen/myosin binding protein C/similar to sarcalumenin.
protein, suggesting that the protein is an isoform of titin. This polypeptide has been reported to be an elastic portion of titin (Matsuura, Kimura, Ohtsuka, & Maruyama, 1991). Four myosin family members, myosin heavy chain 1 (MYH1), myosin heavy chain IIa (MYHIIa), myosin heavy chain 7 (MYH7) and myosin 3-like protein, were also identified in this band with very high score and sequence coverage and number of unique peptides (Table 1), probably as cross-linked or aggregated polymers of myosin. The AHNAK nucleoprotein isoform 1 protein also co-migrates with this 1200 kDa band but showed in very low quality (0.9% sequence coverage and 3 unique peptides). Nebulin in band 5 was confirmed as intact nebulin (773 kDa), and as a major protein in band 5, which had the highest score (8473) and sequence coverage (21.8%) and 160 unique peptides (Table 1). Some myosin super-family members also co-migrated with intact nebulin. This band also contained titin fragments but with lower quality (sequence coverage 1.7%). In the present study, although bands 5 and 6 appeared at similar level in the gel, band 6 (at 28 days post mortem) was identified to consist of only two myosin members, MYH1 and myosin-2 (MYH2). Intact nebulin and titin identified in band 5 were not detected in band 6, suggesting that these proteins were completely degraded at 28 days of post mortem
storage. The rapid degradation of nebulin during post mortem ageing has been reported by a number of other researchers (Geesink & Koohmaraie, 1999; Watanabe & Devine, 1996). Similarly, myosin heavy chain IIa, MYH7 and myosin 3-like protein were not identified in band 6. It showed that the intensity of band 6 at 28 days was greater than that at early post mortem ageing samples. The presence of this band as a large molecule band could be a result of aggregation of MYH1 and MYH2 or the cross-linking of these two myosin heavy chains (Fig. 1). Titin and nebulin are giant structural proteins and abundant in skeletal muscle. Both are thought to act as a ‘‘molecular ruler’’ for the developing myofibril. Degradation of these proteins was documented to be correlated to post mortem tenderization (Huff Lonergan et al., 2010) as observed in the present study. As shown in Fig. 2, intact titin (B1) and intact nebulin (B5) present abundant quantity at day ‘‘0’’ post mortem, they were degraded very fast and hardly which detected at day 7 post mortem storage. As important structural proteins in muscle, the breakdown of these proteins could translate to tremendous changes in the structure of muscle myofibrils. In our previous paper, we hypothesised that the breakdown in meat structure, including these structural proteins, could improve the waterholding capacity of meat with aging (Farouk et al., 2012).
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Table 1 LC–MS–MS identified proteins from 17 bands separated by 1D SDS–PAGE from bull M. longissimus dorsi (LD) muscle during post mortem storage. Band
Accession
Protein name
Score
Sequence coverage (%)
No. of unique peptides
1 2 3 4
gi|297471578 gi|297471578 gi|297471578 gi|297465038 gi|41386691 gi|261245063 gi|41386711 gi|297462470 gi|297464894 gi|119887683 gi|297471578 gi|41386691 gi|261245063 gi|41386711 gi|297462470 gi|75055812 gi|41386691 gi|41386711 gi|75055812 gi|119887683 gi|41386691 gi|75055812 gi|297471578 gi|41386711 gi|297459506 gi|119887683 gi|297462470 gi|297460708 gi|119887683 gi|297465038 gi|75055812 gi|41386691 gi|41386711 gi|297459506 gi|296477710 gi|329663701 gi|520 gi|41386691 gi|41386711 gi|119887683 gi|194666432 gi|194666432 gi|41386691 gi|297489712 gi|297472245 gi|75055812 gi|41386691 gi|329663986 gi|41386711 gi|297459506 gi|300794839 gi|84000245 gi|296472512 gi|41386691 gi|261245063 gi|41386711 gi|297489712 gi|300794727 gi|75055812 gi|41386691 gi|329663986 gi|160425243 gi|300794727 gi|297479068 gi|41386711 gi|296472512 gi|160425243 gi|41386691 gi|75055812 gi|41386711 gi|300794727 gi|75055812 gi|41386691 gi|41386711
Titin [Bos taurus] Titin [Bos taurus] Titin [Bos taurus] Titin [Bos taurus] Myosin, heavy chain 1, skeletal muscle, adult Myosin heavy chain IIa Myosin, heavy chain 7, cardiac muscle, beta Myosin-3-like Low quality protein AHNAK nucleoprotein isoform 1 Nebulin [Bos taurus] Low quality protein titin [Bos taurus] Myosin, heavy chain 1, skeletal muscle, adult Myosin heavy chain IIa [Bos taurus] Myosin, heavy chain 7, cardiac muscle, beta Low quality protein myosin-3-like [Bos taurus] Myosin-2 (Myosin heavy chain 2) myosin, heavy chain 1, skeletal muscle, adult [Bos taurus] Myosin, heavy chain 7, cardiac muscle, beta [Bos taurus] Myosin-2 (Myosin heavy chain 2) Low quality protein nebulin [Bos taurus] Myosin, heavy chain 1, skeletal muscle, adult Myosin-2 (Myosin heavy chain 2) Low quality protein titin [Bos taurus] Myosin, heavy chain 7 Myosin, heavy polypeptide 6, cardiac muscle, alpha-like Low quality protein nebulin [Bos taurus] Low quality protein myosin-3-like [Bos taurus] Low quality protein epiplakin 1 [Bos taurus] Nebulin [Bos taurus] low quality protein titin [Bos taurus] Myosin-2 (Myosin heavy chain 2) Myosin, heavy chain 1, skeletal muscle, adult Myosin, heavy chain 7, cardiac muscle, beta Myosin, heavy polypeptide 6, cardiac muscle, alpha-like Low quality protein ryanodine receptor 2 like isoform Low quality protein cytoplasmic dynein 1 heavy chain 1 Low quality protein b-lactoglobulin [Bos taurus] Myosin, heavy chain 1, skeletal muscle, adult Low quality protein myosin heavy chain 7, cardiac muscle Low quality protein nebulin [Bos taurus] Similar to gamma filamin isoform 3 [Bos taurus] Similar to gamma filamin isoform 3 [Bos taurus] Low quality protein myosin, heavy chain 1, skeletal muscle Myomesin 1, 185 kDa isoform 1 [Bos taurus] Myomesin family, member 3 [Bos taurus] Myosin-2 (Myosin heavy chain 2) Myosin, heavy chain 1, skeletal muscle, adult [Bos taurus] myosin-8 [Bos taurus] myosin, heavy chain 7, cardiac muscle, beta [Bos taurus] Myosin, heavy polypeptide 6, cardiac muscle, alpha-like Synemin [Bos taurus] Low quality protein myomesin (M-protein) 2 [Bos taurus] Myomesin (M-protein) 2, 165 kDa [Bos taurus] myosin, heavy chain 1, skeletal muscle, adult [Bos taurus] Myosin heavy chain IIa [Bos taurus] Myosin, heavy chain 7, cardiac muscle, beta [Bos taurus] Myomesin 1, 185 kDa isoform 1 [Bos taurus] Glycogen debranching enzyme [Bos taurus] Myosin-2 (Myosin heavy chain 2) Myosin, heavy chain 1, skeletal muscle, adult Myosin-8 [Bos taurus] Myosin binding protein C, slow type Glycogen debranching enzyme [Bos taurus] Myosin, heavy polypeptide 6, cardiac muscle, alpha-like Myosin, heavy chain 7, cardiac muscle, beta [Bos taurus] Low quality protein myomesin (M-protein) 2, 165 kDa Myosin binding protein C, slow type [Bos taurus] Myosin, heavy chain 1, skeletal muscle, adult Myosin-2 (Myosin heavy chain 2) Myosin, heavy chain 7, cardiac muscle, beta [Bos taurus] Low quality protein glycogen debranching enzyme Myosin-2 (Myosin heavy chain 2) Myosin, heavy chain 1, skeletal muscle, adult [Bos taurus] Low quality protein myosin, heavy chain 7
28978.5 16898.1 22,564 17158.7 3592.3 3532.9 1912.3 1155.1 168.8 8473.1 2097.2 2066.7 1984.6 1067.9 707.4 1291.7 1070.1 913.4 626.7 331.7 2662.3 2296.2 1923.8 1686.2 1559.5 1460.5 627.8 150.4 6053 2463.2 1857.5 1798.9 788 730.1 724.4 435.5 368.9 940.2 518.9 501.4 2756.6 3927.1 338.9 6051.1 2645.3 2276.5 1901.7 1742.3 1716.7 1511.5 958.6 355.4 2574.1 2228.7 2083 1252.9 1240.3 1156.1 5178.3 4748.7 3321.9 3291.3 1725.6 1693.8 1643.7 296 3651.2 1232.8 1209.9 1174.4 340.4 980.4 908.5 555.9
22.4 12.2 15.5 13.6 37.3 35.6 21.6 10.8 0.9 28.5 1.7 21.8 20.1 12.8 7.2 13.9 11.4 9.3 6 1.1 29.5 24.5 1.6 18.8 17.6 5.9 7.5 1.1 20.2 2.3 21.2 21.1 9 8.3 3.8 2.8 44.4 10.4 6 2.7 20.1 30.4 4 67.1 41.5 25.8 20.2 19 18.7 16.7 14.4 10.3 59.1 25.2 23.5 13.7 17.4 16.9 45.6 40.7 29.3 49.6 25.7 14.2 14.2 7.5 53.7 14.2 14.5 13.7 6.1 14.5 13.4 7.1
559 314 408 344 65 62 36 21 3 160 40 37 34 21 13 23 19 18 12 7 48 42 38 33 30 29 14 4 117 54 33 33 15 13 12 9 6 18 9 9 47 61 7 106 45 42 36 33 33 29 18 7 41 43 41 25 21 21 87 81 56 58 32 30 31 5 65 22 21 22 6 18 16 9
5
6 7
8
9
10
11 12 13
14
15
16
17
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G. Wu et al. / Food Chemistry 150 (2014) 137–144 Table 1 (continued) Accession
Protein name
Score
Sequence coverage (%)
No. of unique peptides
gi|219804724 gi|160425243 gi|119916692
Low quality protein collagen, type VI, alpha 1 [Bos taurus] Low quality protein myosin binding protein C, slow type Low quality protein sarcalumenin [Bos taurus]
300.5 290.5 239.5
9.8 9.5 15.6
6 6 5
B2
B1
60 40 20
160
140
100
100
120 100 80 60 40 20
0
0
2
100
0
7
21
80 60 40 20
2
Post mortem (day)
0
2
7
21 28
Post mortem (day)
60 40 20
2
0
Post mortem (day)
B12
B14 120
120
100
100 80 60 40 20 7
21 28
Post mortem (day)
2
Post mortem (day)
140
0
0 0
80
0 0
28
Relative Qty (%)
100
Relative Qty (%)
Relative Qty (%)
120
20
20
B11
120
40
40
Post mortem (day)
B10
60
60
0 2
Post mortem (day)
80
80
0
7
Relative Qty (%)
80
B8 120
Relative Qty (%)
100
Relative Qty (%)
Relative Qty (%)
120
B5 120
Relative Qty (%)
Band
80 60 40 20 0 0
2
7
21 28
Post mortem (day)
Fig. 2. Quantity changes of the most interesting bands shown in Fig. 1 during post mortem period. Quantities (Qty) of these bands were analysed by Quantity One software (4.6.5) from Bio-Rad. The identification of these bands is shown in Table 1. B = the abbreviation of band.
3.2. Protein bands with molecular weight between 600 and 230 kDa: Bands 7–12 Band 7 consists of MYH7, MYH2 and a little quality fragment of nebulin (1.1% sequence coverage). The intensity of this band did not show obvious change during post mortem ageing of muscle/ meat (Fig. 1). Band 8 mainly contained MYH1 and MYH2, together with MYH7 and myosin heavy polypeptide 6 (Myh6). Myosin 3-like protein existed in this band as well. Incidences of titin and nebulin fragments and epiplakin 1 in this band were only in very low quality (Table 1). Epiplakin is a member of the plakin family. There is a dearth of information about its role in beef muscle and tenderness (Fujiwara et al., 2001). The quantity of band 8 exhibited a clear decline with ageing and it was hardly detectable after 7 days post mortem (Figs. 1 and 2). A major degradation product of nebulin was confirmed in band 9 (21 days post mortem sample) which displayed the top rank (highest score with highest unique peptides and sequence coverage) among the components in the band. In addition, several myosin family proteins, MYH1, MYH2, MYH7 and Myh6, were present. Interestingly, beta lactoglobulin was also identified in this band.
This small protein (18 kDa) presenting at the large molecule position on the SDS–PAGE could be a crosslinked product of b-lactoglobulin with other structural muscle proteins (Eissa, Puhl, Kadla, & Khan, 2006). Although fragment of titin was detected in this band, it only occurred in very low quality (sequence coverage 2.3%). The other two proteins identified in this band were ryanodine receptor 2 (cardiac)-like isoform 2 and cytoplasmic dynein 1 heavy chain 1. They also existed in relative lower quality. The relevance of these two proteins in meat quality has not yet been elucidated. The composition of bands 7–9 has not previously been reported, probably due to their low protein content and the limited sensitivity of detection by the Coomassie stain commonly used in SDS–PAGE gels. Band 10, which appeared in the samples of 0 and 2 days and hardly detected after 7 days post mortem, apparently degraded with post mortem ageing (Figs. 1 and 2). Early research on striated muscle chemistry described this band as co-migrating with a dimer of myosin heavy chain (Wang, 1982). This band contained MYH1, MYH7 and a low quality fragment of nebulin (Table 1). Filamin was identified in bands 11 and 12 (Fig. 1). There are several different isoforms of intact filamin. Band 11 was shown as a
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Table 2 The 26 beef muscle/meat proteins (>100 kDa) identified by LC–MS/MS from bull M. longissimus dorsi (LD) muscle during post mortem storage.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Accession
Protein name
MW (kDa)
pI
gi|297471578 gi|297465038 gi|41386691 gi|261245063 gi|41386711 gi|297462470 gi|297464894 gi|119887683 gi|75055812 gi|297459506 gi|297479068 gi|297460708 gi|296477710 gi|329663701 gi|520 gi|194666432 gi|297489712 gi|297472245 gi|329663986 gi|300794839 gi|84000245 gi|296472512 gi|300794727 gi|160425243 gi|219804724 gi|119916692
Titin [Bos taurus] Titin isoform [Bos taurus] Myosin, heavy chain 1, skeletal muscle, adult Myosin heavy chain IIa Myosin, heavy chain 7, cardiac muscle, beta Myosin-3-like AHNAK nucleoprotein isoform 1 [Bos taurus] Nebulin [Bos taurus] Myosin-2 (Myosin heavy chain 2) Myosin, heavy polypeptide 6, cardiac muscle Myosin, heavy polypeptide 6, cardiac muscle, alpha-like Epiplakin 1 [Bos taurus] Ryanodine receptor 2 (cardiac)-like isoform 2 [Bos taurus] Cytoplasmic dynein 1 heavy chain 1 [Bos taurus] b-lactoglobulin [Bos taurus] Similar to gamma filamin isoform 3 [Bos taurus] Myomesin 1, 185 kDa isoform 1 [Bos taurus] Myomesin family, member 3 [Bos taurus] Myosin-8 [Bos taurus] Synemin [Bos taurus] Myomesin (M-protein) 2, 165 kDa [Bos taurus] Myomesin (M-protein) 2, 165 kDa [Bos taurus] Glycogen debranching enzyme [Bos taurus] Myosin binding protein C, slow type [Bos taurus] Collagen, type VI, alpha 1 [Bos taurus] Similar to sarcalumenin (predicted) [Bos taurus]
3713.5 3713.5 222.9 223.2 223.1 223.9 500.7 772.9 223.2 223.7 222.7 380.3 565 531.6 18.3 287.2 174.7 159.9 222.6 170.7 104 104.2 174 133.9 108.6 54.4
6.0 6.0 5.5 5.5 5.5 5.5 5.6 9.6 5.5 5.5 5.5 5.6 5.0 6.0 4.7 5.6 6.1 5.5 5.5 4.8 7.0 7.2 6.3 5.5 5.1 6.2
singlet protein in all animal samples at 0 time point. This band has previously been identified as intact filamin by Western blots and immunocytochemistry (Huff-Lonergan, Mitsuhashi, Beekman, et al., 1996; Van Der Ven et al., 2000). After 7 days post mortem band 11 appeared to migrate as doublet bands, an intact filamin and a degraded filamin product (B12). Band 12 contained two proteins, similar to gamma filamin isoform 3 and MYH1 (Table 1). The MYH1 in this band was present in lower quality (Table 1). This filamin form is thought to be a degradation product of intact filamin from band 11 (Huff Lonergan et al., 2010; Huff-Lonergan, Mitsuhashi, Beekman, et al., 1996). Previous research on filamin in mammalian muscle has classed it conflictingly as both absent (Wang, Ash, & Singer, 1975; Wang & Singer, 1977) and present (Chiang & Greaser, 2000; Van Der Ven et al., 2000) in striated muscle. Our result is in agreement with the later studies. Moreover, it is clearly showed that intact filamin (B11, Fig. 2) degraded with aging time and its degradation product increased after 7 days post mortem (B12, Fig. 2). 3.3. Protein bands with molecular weight between 200 and 100 kDa: Bands 13–17 There were five strong bands between myosin heavy chain (MHC) and the 121 kDa molecular weight marker on the gel. The top band among these (band 13) was identified as a mixture of myomesin family, myosin family and synemin proteins. In the myomesin family, there are three members, namely myomesin1 (185 kDa isoform1), myomesin 3 (myomesin family member 3) and myomesin-2. Myomesin-2 (accession number gi|84000245) is regarded as a cluster member of myomesin (M protein) 2, 165 kDa (Table 1). It displayed in low quality (Table 1). The myosin family members in band 13 consisted of MYH2, MYH1, MYH7, myosin-8 and Myh6, which are possibly fragments of myosin heavy chain. Among the identified proteins, myomesin 1 and myomesin 3 had the highest score and sequence coverage (Table 1). Myomesin 2 was mostly revealed in band 14. It exhibited the highest score (2574) and sequence coverage (59.1%). In addition, band 14 consisted of myomesin family members 1, fragments of myosin
family members (MYH1, MYHIIa and MYH7) and glycogen debranching enzyme. Quantity of this band changed obviously with aging time. The highest quantity of this band was shown at 2 days post mortem. After this time point, the quantity decreased with the storage time increase (B14, Fig. 2). The change of this band, especially the myomesin 2, could play an important role to meat quality. Band 15 was mainly composed of fragments of myosin family members, including MYH2, MYH1, MYH7, myosin-8 and Myh6. Furthermore, glycogen debranching enzyme and myosin binding protein C occurred in this band. Although myomesin (M protein) 2 were detected in this band as well, it only showed in low quality (Table 1). Myosin binding protein C (slow type) was identified predominantly in band 16 along with fragments of MYH1, MYH2, MYH7 and glycogen debranching enzyme. Of these proteins, myosin binding protein C demonstrated the highest score of 3651 with sequence coverage of 53.7%. Band 17 was identified as a mixture of proteins. Besides the myosin class proteins (MYH2, MYH1, MYH7) and low quality myosin binding protein C, this band contained two other proteins, collagen type VI (alpha 1) and a protein described as similar to sarcalumenin (Tables 1 and 2). Sarcalumenin is a calcium-binding protein (Jiao et al., 2009; Yoshida et al., 2005). There is lack of information regarding the roles of this protein in meat quality. In the present study, twenty-six individual large proteins were identified in the seventeen bands in the gel (Table 2). Myosin family members, especially MYH1, MYH2 and MYH7, were shown to migrate in numerous bands. Some of these proteins appeared to co-migrate with very large proteins, i.e. 1200 kDa titin band (B4) and nebulin band (B5). Whether this reflects cross-linking or non-covalent interactions between these myosins and titin/nebulin is unknown. Myosin molecules seem to polymerize to form large protein aggregates or polymers in pre-rigor muscle samples. Some of these larger myosin molecules (band 8 and 10) gradually decrease during ageing. The migration of MYH1 and MYH2 in band 6 (at 28 days post mortem) is apparent that it was in a cross-linked or aggregated form (600–800 kDa). Myosin identified in bands at higher molecular weights than myosin heavy chain (MHC) on the gel could be due to cross-linking or aggregation. Similar results
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were reported in a study of myosin degradation in chicken meat (Ikeuchi et al., 2001). This may reflect interaction of the long tail region of myosin with other proteins or with other regions of myosin chains through disulfide bonds, or to the cross-linking activity of transglutaminase in muscle (Ahhmed et al., 2009; Eligula et al., 1998; Ooizumi & Xiong, 2004). Conventional SDS–PAGE is difficult to separate the myosin heavy chain isoforms as these protein subunits are the most abundant in skeletal muscle with close molecular weight and isoelectric points. Some proteins, such as glycogen debranching enzyme and myosin binding protein C, occurred in more than one band, which could be attributed to partial proteolysis or the existence of multiple isoforms of these proteins. Three myomesin family members, myomesin 1, myomesin 2 and myomesin 3 were identified in the present study. There has been very limited research on myomesins in relation to meat quality. This could because myomesins consist mainly of immunoglobulin and fibronectin domain types, and were regarded as members of the immunoglobulin superfamily (Tskhovrebova & Trinick, 2012). Researches have demonstrated these proteins are mostly located in the M-band of sarcomere, and were thought to be involved in anchoring the thick filaments of the sarcomere (myosin) to other filaments (titin), stabilizing and aligning the structure of muscle (Schoenauer et al., 2008). Although specific function of these myomesins is still not completely understood, it has been shown that it is present from the earliest stages of skeletal muscle tissue formation. Based on myomesin’s specific location in the sarcomere of myofibril and muscle structure (Tskhovrebova & Trinick, 2012), one would expect these proteins to play a role in meat quality, although little is known about these proteins in this regard. It has been suggested that myomesin may function as a molecular spring that protects the sarcomere and keeps it stable during intense or sustained stretching (Schoenauer et al., 2005). In a previous report (Anderson, 2011), it was suggested that myomesin 2 could be a potential indicator of post mortem tenderness. Our results also showed a decrease in the quantity of this protein (B14) with post mortem ageing (Fig. 2), but further study is needed to determine its role in meat tenderization. 4. Conclusions A total of 26 large bovine muscle proteins (>100 kDa) were separated using SDS–PAGE and identified by LC–MS/MS. These proteins included intact titin, nebulin, filamin and their degradation products. We confirmed degradation of these three proteins during post mortem ageing. In addition, other large proteins, including myosins, myomesins, synemin, myosin binding protein C, ryanodine receptor 2 and others were identified in the muscle/meat samples. Three myomesin members were present in the skeletal muscles and could play a potential role in meat tenderization, since their concentration decreased during post mortem ageing. A number of myosin super-family members were revealed on SDS–PAGE, and shown to spread broadly in the gel in the forms of crosslinked/aggregated proteins and fragments. Further studies are needed to determine the roles of myomesins, myosins in meat tenderization process and changes of myosins and other structure proteins with aging time. 5. Implications The outcomes reported in the present study have the following implications within the parameters of the study: 1. The use of 1D SDS–PAGE in conjunction with LC–MS/MS to identify large muscle structural proteins and the changes of these proteins undergo very early post mortem (30 min) was demonstrated.
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2. The changes observed highlighted the important role of these large proteins in the early tenderization of meat post mortem and supports the recently reported hypothesis of pH compartmentalised tenderness in meat (Lomiwes, Farouk, Wu, et al., 2014) 3. The composition of band 7–9 is reported for the first time in the present study. This will help in the understanding of the biophysical changes occurring in muscles/meat early post mortem and the effect of the changes on meat quality. 4. The migration of myosin heavy chain isoforms with associated large molecular weight proteins such as titin very early post mortem seem to suggest that the breakdown of muscle proteins and consequently improvement in tenderness is initiated at the A-band region of the muscle cell sarcomere rather than the I-band where the z-disk is located as is currently widely accepted. This hypothesis needs to be investigated further. 5. There are structural proteins changes observed that require further studies to be fully explained in relation to meat quality.
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