Proteomic approach for the detection of chicken mechanically recovered meat
I. Surowiec, K.M. Koistinen
1 , P.D. Fraser, P.M. Bramley School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK a b s t r a c t a r t i c l e i n f o Article history: Received 24 November 2010 Received in revised form 5 April 2011 Accepted 6 April 2011 Keywords: Chicken meat analysis Mechanically recovered meat Hand deboned meat OFF-GEL electrophoresis Nano-LC-MS/MS Protein identication Mechanically recovered meat is cheaper than rawmeat and thus has been incorporated into many meat-derived products. EU regulations exclude mechanically recovered meat from the denition of meat; as a consequence analytical procedures are neededto differentiate it fromhand-deboned meat. The present pilot study has utilized a proteomic approach to nd potential markers for the detection of chicken mechanically recovered meat. Intact proteins were extracted fromrawmeat and then analyzed with OFF-GEL electrophoresis followed by SDS-PAGE andidenticationof potential markers bynano-LC-MS/MS. It was shownthat it is possibletoextract, separate and identify key proteins from processed meat material. Potential chicken mechanically recovered meat markers hemoglobin subunits and those similar to myosin-binding protein C were also identied. 2011 Elsevier Ltd. All rights reserved. 1. Introduction Mechanically recovered meat (MRM) is obtained by recovering residual raw meat from animal bones or poultry carcasses from which the bulk of the meat has beenremoved. This is typically achievedusing a machine that applies high pressure or shear forces to the animal bones or poultry carcasses (BMMA, 1991). Such machines allow most of the residual meat, which would otherwise be difcult or uneconomical to obtain, to be recovered. MRMhas the appearance of nely comminuted meat and is used in a wide range of meat products, as an inexpensive source of meat. Although MRM has a similar chemical composition to authentic or hand de-boned meat (HDM), consumers see MRM as a cheap, inferior material and treat it with suspicion. This has led to the exclusion of MRM from the EU denition of meat (Directive No 101/ 2001). In doing so, clear and separate labeling of MRM in products is required. Therefore there is a need for reliable analytical methods that can differentiate MRM from HDM. Numerous approaches have been employed to differentiate MRM from HDM. Histological approaches have been developed that exploit changes in meat properties arising during the mechanical production process whichafter the appropriate staining can be visualized under the microscope (Pickering, Evans, Hargin, & Stewart, 1995a; Tremlova, Sarha, Pospiech, Buchtova, &Randulova, 2006). Although this method is so far the most commonly used in MRM detection, it does not give reliable results and is non quantitative. In addition, it is time consuming and requires considerable expertise. These properties preclude the methodology as a robust, routine mode of analysis for the food industry. Another approach is based on the assumption that during MRM production uids from bone structures are released into the meat derived material. These uids can be immunologically different from meat itself, and even from the residual blood that is always found in hand-debonedmeat. Potential MRMspecic polyclonal antibodies were obtained by raising them against a low molecular weight fraction of chicken bone marrow, and then used to screen MRM, HDM and MRM- HDM mixtures using ELISA based assays (Pickering, Grifn, Smethurst, Hargin, & Stewart, 1995b). Results were, however, equivocal, mainly due to the lowselectivity of the procedure which was highly inuenced by residual blood, skin and other tissues. This approach indicated that immunological tests cannot be used for MRM detection, unless further optimization of the procedure is achieved, for example by production of more specic antibodies. Another approach for MRMidentication is based on the application of protein analysis methods. It is based on the assumption that some bone proteins, not found in raw meat, can be released into the nal product during MRMproduction or relative quantities of some proteins can differ between both kinds of material. Electrophoretic techniques have been used to separate meat proteins including SDS-PAGE (Field, Sanchez, Ji, Chang, & Smith, 1978; Savage, Richardson, Jolley, Hargin, & Stewart, 1995), capillary gel electrophoresis (Day & Brown, 2001) or isoelectric focusing followed by multivariate data analysis (Skarpeid, Moe, & Indahl, 2001). Differences in the relative concentrations of several proteins were observed, with hemoglobin content higher in marrowthan in meat, and hence also higher in MRMthan HDM. On the other hand, HDMwas characterized by higher amounts of actin, myosin andmyoglobin. Someother distinct proteinbands werealsonoticed, but Meat Science 89 (2011) 233237 Corresponding author at: Tel.: +44 1784443555; fax: +44 1784414224. E-mail address: P.Bramley@rhul.ac.uk (P.M. Bramley). 1 Current address: Zora Biosciences Oy, Biologinkuja 1, FI-02150 Espoo, Finland. 0309-1740/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.04.004 Contents lists available at ScienceDirect Meat Science j our nal homepage: www. el sevi er. com/ l ocat e/ meat sci they were not identied. Since very limited numbers of samples were included in these studies and no information about repeatability of the results was given, the approach needs further validation. There are fewaspects to consider when applying methods of protein analysis for meat samples. When the whole proteome analysis is concerned, 1-D gel electrophoresis cannot provide sufcient resolution and less abundant, but potentially signicant, proteins can be missed when SDS-PAGE alone is used. Therefore proteomics relies heavily on2- Dgel electrophoresis, whichis laborious. An alternative approach to 2-D gel electrophoresis is OFF-GEL electrophoresis, where proteins after separation according to their pI values, are recovered fromsolution and can be directly used for SDS-PAGE separation, enzyme digestion, crystallization or mass spectrometry (Michel et al., 2003). The OFF- GEL approach is a good alternative to classical 2-D gel electrophoresis and has successfully been used in protein purication in E. coli extracts (Ros et al., 2002) and proteome analysis of body uids (Burgess et al., 2006; Heller et al., 2005). In the current pilot study, OFF-GEL electrophoresis, followed by SDS-PAGE and nano-LC-MS/MS identication of proteins, was applied to identify biomarkers that could be potentially used for the detection of chicken MRM in meat products. 2. Material and methods 2.1. Reagents and standards Hand-deboned and mechanically recovered chicken meat samples were obtained from Leatherhead Food International (Leatherhead, UK) and prepared according to the Regulation (EC) No 853/2004. Samples were produced on a commercial scale using batches of 10 to 20 kg of chickens. Hand deboned meat material was removed and the remainder used to prepare MRM. The apparatus used was a Lima (RM500) set at 18 bars and 3 mm. The material was placed onto large stainless steel trays at a depthof 1 in. The pooledsample was prepared fromat least three trays. After manufacturing, 28 kg of eachmeat product were frozenat once and delivered to Royal Holloway, University of London, where they were stored at 20 C until further treatment. Small pieces of samples were taken fromdifferent parts of the large blocks, homogenized with Waring Commercial Laboratory Blender (Waring Products, Torrington, UK) and storedat 80 Cuntil use. Three samples of eachkindof meat were used. The following chemicals were used: sodium dodecyl sulfate (app. 99%), ammoniumbicarbonate (minimum99.0%), urea (98+%), thiourea (ACS reagent), 3-[(3-cholamidopropyl)dimethylammonio]-1-propane- sulfonate (CHAPS) (98% TLC), dithiothreitol (DTT) (for electrophore- sis, 99%), glycerol for molecular biology (minimum 99%), bromophenol blue sodium salt (for molecular biology, for electrophoresis) and ampholyte 310 for isoelectric focusing from Aldrich (Steinheim, Germany), water (for HPLC) and TRIS (molecular biology grade, minimum 99%) from BDH (Poole, England), triuoroacetic acid (TFA) from Fluka (Buchs, Switzerland), formic acid (Super Purity Solvent) from Romile Pure Chemistry (Cambridge, UK), acetonitrile (HPLC gradient grade) from J. T. Baker (Deventer, Holland), trypsin, modied (sequencing grade) from Promega (Madison, WI, USA) and hydro- chloric acid (analytical grade) from Fischer Scientic (Loughborough, UK). 2.2. Preparation of protein extracts from meat samples Immediately prior to extraction, samples were allowed to thaw at room temperature. To raw meat material (1 g), 3 mL of extraction buffer (7 M urea, 2 M thiourea, 2% CHAPS and 10 mM DTT) were added and samples were shaken gently for 30 min at RT. Then, samples were centrifuged at 3000 g for 10 min, two 1 mL aliquots of the supernatant were taken and centrifuged at 16,000 g for 10 min. Supernatants were combined, and proteins precipitated with 100% ice cold acetone (10 mL). Samples were then incubated overnight at 20 C. Next day, the protein pellet was washed three times with ice cold acetone and centrifuged at 16,000 g for 10 min, the acetone decanted and sample dried in vacuum centrifugal evaporator (Genevac EZ-2 Evaporator from Genevac Inc (New York, USA)) for 15 min. The dry pellet was dissolved in 1 mL of OFFGEL buffer containing 7 M urea, 2 M thiourea, 65 mM DTT, 5% glycerol and 0.5% (v/v) of ampholytes (pH 3.010.0). Protein concentration was measured using the Bio-Rad Protein Assay Dye reagent. A total of 500 g protein was loaded into each IPG strip (Immobiline DryStrip) from GE Healthcare (Uppsala, Sweden). 2.3. OFFGEL electrophoresis (OGE) TheOGEfractionationwas performedas previously described(Heller et al., 2005) using a 3100 OFFGEL Fractionator fromAgilent Technologies (Morges, Switzerland). EachOGE fraction contained proteins spanning a pI range of approximately 0.3 units, as can be assumed from the conguration of the OGE device: 24 equal wells corresponding to 24 fractions obtained for each sample distributed over the pH 310 range. IPGstrips (24 cm, pH3.010.0) were rehydratedina solutioncontaining 7 M urea, 2 M thiourea, 65 mM DTT, 0.5% ampholytes and 5% (v/v) glycerol. A 24-well device was then placed on the rehydrated IPG and 50 L of sample was loaded in each well across the whole strip. The separation was carried on at constant current (50 A) for 64 kVh with maximum voltage equal to 8 kV. Fractions were recovered from each of the wells andrunonSDS-PAGE gels. Eachtype of meat (HDMand MRM) was analyzed three times to check the repeatability of the results. 2.4. SDS-PAGE and in-gel digestion Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS- PAGE) was run in the Mini-PROTEAN 3 from BioRad Laboratories (Hercules, CA, USA) according to manufacturer's instructions. 10 L of solution made from 8 L off OFFGEL fraction and 2 L of sample loading buffer (0.25 M TrisHCl, pH 6, 8, 10% SDS, 50% glycerol, 0.05% bromophenol blue) was loaded onto a 12% gel. Gels were stained with a silver stain kit ProteoSilver from Sigma-Aldrich (Steinheim, Germany) according to the manufacturer's protocol. In-gel digestion was performed according to Koistinen et al. (2002). Peptides were reconstituted in 65 L of 0.1% TFA water solution and 60 L were injected into the LC column. 2.5. Protein identication with nano-LC-MS/MS analysis Peptides were separated using Ultimate/Famos nano LC system from LC Packings (Amsterdam, The Netherlands). The sample was loaded onto a 200 m i.d. 5 mm PS-DVB monolithic trap column from Dionex (Sunnyvale, CA, USA) with a ow rate of 10 L/min of 0.1% TFA for 30 min. After preconcentration, the trap column was automatically switched in-line with the 100 m i.d. 50 mm PS-DVB monolithic analytical column from Dionex and the peptides were eluted with a linear gradient starting from 95% eluent A (0.1% formic acid in water) to 40% of eluent B (0.1% formic acid in ACN) in 40 or 120 min, the ow rate being 200 nL/min. The LC was connected to mass spectrometer with a nanoES ion source from Protana (Odense, Denmark) using 10 m PicoTip from New Objective (Woburn, MA, USA). The positive TOF mass spectra were recorded on a QSTAR Pulsar i hybrid quadrupole TOF instrument from Applied Biosystems (Foster City, CA, USA) using information-dependent acquisition (IDA). TOF MS survey scan was recorded for mass range m/z 400 to 1600 followed by MS/MS scans of the two most intense peaks. Typical ion spray voltage was in the range of 2.0 to 2.4 kV and N 2 was used as collision gas. Other source parameters and spray position were optimized with the tryptic digest of bovine serum albumin. 234 I. Surowiec et al. / Meat Science 89 (2011) 233237 2.6. Database search and protein identication An automated spectral processing, peak list generation and database search from raw data acquired on a QTOF instrument was performed using Analyst QS v1.1 and the MASCOT Search v1.6b13 script for Analyst QS fromAppliedBiosystems, incombinationwiththe MASCOT interface. Identications were carried out using the NCBI non-redundant protein database (Metazoa). Mascot was used with a precursor mass error of 1.2 Da, fragment ion mass tolerance of 0.6, unknown charge state and oxidized methionine as a variable modication. Matches of MS/MS spectra against sequences in the databases were also veried manually. 3. Results and discussion 3.1. OFF-GEL electrophoresis and SDS-PAGE separation Proteins extracted from chicken meat were rst analyzed with SDS- PAGE alone, but the separation was not sufcient to reliably identify proteins that differ between HDM and MRM, because of the low resolution obtained with the SDS-PAGE approach (data not shown). To increase resolution, proteins extracted from chicken meat were rst fractionated by OGE according to their pI's using a pH gradient ranging from 3.0 to 10.0. The 24 fractions obtained from OGE were then separated by SDS-PAGE. Fig. 1 shows silver-stained SDS-PAGE gels of several OGE fractions for one MRM(M) and one HDM(H) meat sample (the lower the number of the fraction, the lower the pI of the proteins). Only gels from which protein bands for further LC-MS analysis were taken are shown. The quality of OGE fractionation can be clearly seen, with some bands represented in multiple fractions and others concentrated in one or two fractions, with some bands including only one protein, others two or more (Table 1). Three replicates of each sample revealed that fractionation of the samples was highly reproduc- ible comparable protein patterns of respective OGE fractions were obtained on SDS-PAGE gels, showing that OFF-GEL electrophoresis is a reproducible method for separation of meat proteins. Visual comparison of SDS-PAGE gels after OGE showed that different protein patterns were obtained for MRM and HDM samples. As seen in Fig. 1, the most visible differences between hand-deboned and MRM samples were observed for fractions 19 and 20, with an intense protein band at around 15 kDa being present predominantly in MRM extract (bands 5 and 6 in Fig. 1C). Other reproducible differences were obtained for fractions 11 and 12, with a band of app 130 kDa more intense in the MRM extract than in the HDM one (bands 3 and 4 at Fig. 1B). These bands, together with bands from fractions 7 and 8 with an approximate mass of 30 kDa (Fig. 1A, band1 more intense inhand-deboned meat and band 2 more visible in the MRMsample), were chosen for further nano- LC-MS/MS analysis to analyze the possibility of identication of main and minor proteins from chicken meat after their OGE and SDS-PAGE fractionation and to identify peptides that could serve as potential biomarkers for MRM detection. These bands were chosen for further consideration after visual inspection of gels as clearly differentiating between the two kinds of meat. Their intensity was also acceptable and henceit was expectedthat theywouldgiveclear signals inLC-MSanalysis. Thereweremorebandswithlower intensitythat couldbeuniquefor MRM or HDM samples as well, they were however not analyzed in this pilot study but will be of potential interest in a subsequent project focused on more detailed analysis of proteomic differences between HDMand MRM. 3.2. LC-MS/MS analysis Results of nano-LC-MS/MS analysis of the bands shown at Fig. 1 are presented in Table 1. Identication of the proteins was reliable at least three peptides were identied for the main proteins, with good score values for both protein and peptide identication. From the results it was concluded that at least two proteins could potentially be used as reliable MRM markers in OFF-GEL+SDS-PAGE analysis of meat samples: one similar to myosin-binding protein C, slow- type (slow MyBP-C) from bands 3 and 4 and hemoglobin subunits from bands 5and6, bothwerefoundinhigher amounts inMRMsamples. Since the most abundant proteins in both bands 1 and 2 were myosin subunits, it was concluded that the pattern obtained could result from a different distribution of these proteins between fractions 7 and 8 and hence they are not reliable markers for differentiating between HDM and MRM samples. Myosin-binding protein C is a muscle sarcomeric protein with Fig. 1. SDS-PAGE gels (AC) of OGE fractions from one hand-deboned and one MRM chicken extract with the marked bands taken for LC-MS/MS analysis; MMRMsample, H HDM sample; numbers according to the fraction order, starting from lowest to highest pH. For detailed description see text. 235 I. Surowiec et al. / Meat Science 89 (2011) 233237 both structural and regulatory roles (Oakley, Hambly, Curmi, & Brown, 2004). However, it is not clear why there is a higher content of slow MyBP-C in mechanically recovered meat. One of the possibilities is that the extra pressure, temperature and shear forces applied during MRM production may have resulted in the release and dispersion of enzymes whose activity could facilitate better MyBP-C recovery. This assumption needs further investigation. Hemoglobin content in MRM at high levels is probably derived from bone marrow, (Field et al., 1978), conrming its applicability as a potential MRM marker. Although, taking into consideration the fact that residual blood can be also found in HDM products, additional markers should be used for reliable MRM detection in commercial samples, for example MyBP-C. To overcome the variation resulting from different sources and conditions applied to obtain commercial meat materials and MRM production methods, ratios of selectedprotein/peptidebiomarkers toother proteins/peptides present in the sample (ex. myosin chains) could be applied. The present results are an improvement on previous procedures (Savage et al., 1995), where hemoglobin was potentially suggested as marker for chicken MRM, but its identication was not conrmed. Additionally, application of the high-resolution approach based on OFF- GEL electrophoresis and SDS-PAGE enabled identication of another potential protein marker for MRM detection, with the whole methodol- ogy being fast and reproducible. Previous works (Day & Brown, 2001; Field et al., 1978; Savage et al., 1995) have suggested that the muscle proteins actin and myosin can be also used for differentiation between MRMandHDMmeat; results inthis study suggest however that observed Table 1 LC-MS/MS analysis of protein bands from SDS-PAGE gels shown in Fig. 1. The AC number refers to database accession number of matching proteins; peptides identied are also indicated along with their m/z values, charge states and ion scores as given by Mascot. Band Identication (score) AC number Peptides (m/z, charge state, score) 1 Myosin light chain 1f; (282) gi|212330 EAFLLFDR, 505.77, (+2), 46 ITLSQVGDIVR, 600.85, (+2), 89 MTEEEVEELMK, 684.31, (+2), 29 MTEEEVEELMK+Oxidation (M), 692.31, (+2), 17 DQGTFEDFVEGLR, 756.87, (+2), 91 KPAAAAAPAPAPAPAPAPAPAKPK, 533.07, (+4), 28 2 Slow skeletal ventricular myosin alkali light chain 3 (Gallus gallus); (478) gi|45384044 EAFSLFDR, 492.74, (+2), 55 EVEFNPASIK, 567.30, (+2), 44 ITYAQCGDVLR, 619.81, (+2), 72 KEVEFNPASIK, 421.23, (+3), 45 ALGQNPTQAEVMK, 693.86, (+2), 69 VEFTPDQIEEFK, 741.37, (+2), 18 DTGTYEDFVEGLR, 751.34, (+2), 77 VFDKEGNGTVMGAELR, 574.96, (+3), 37 TKDTGTYEDFVEGLR, 577.62, (+3), 63 VFDKEGNGTVMGAELR+Oxidation (M), 580.62, (+3), 26 Myosin light chain 3, skeletal muscle isoform (A2 catalytic); (341) gi|55584150 EAFLLFDR, 505.77, (+2), 55 ITLSQVGDIVR, 600.86, (+2), 85 MTEEEVEELMK, 684.32, (+2), 77 MTEEEVEELMK+Oxidation (M), 692.31, (+2), 35 DQGTFEDFVEGLR, 756.86, (+2), 86 Smooth muscle gamma actin; (50) gi|2967678 GYSFVTTAER, 565.78, (+2), 52 3 PREDICTED: similar to Myosin-binding protein C, slow-type (Slow MyBP-C); (595) gi|118082876 LSVDLRPLK, 520.85, (+2), 59 MIEGVAYEVR, 583.81, (+2), 43 ISLALEDQTVR, 622.86, (+2), 65 LEIPITGEPTPK, 647.88, (+2), 42 IAFQYGITDLR, 648.86, (+2), 65 NTENDTIIFIR, 668.36, (+2), 52 NEAGEDSALINIK, 687.37, (+2), 92 DDGNAAITGYTIQK, 733.88, (+2), 44 FMVELADPTVELK+Oxidation (M), 754.40, (+2), 45 NDNATYSVMTTGGQSEAK, 937.45, (+2), 78 4 PREDICTED: similar to Myosin-binding protein C, slow-type (Slow MyBP-C); (260) gi|118082876 FTITGLPTGSK, 561.32, (+2), 40 MIEGVAYEVR, 583.80, (+2), 39 ISLALEDQTVR, 622.86, (+2), 56 LEIPITGEPTPK, 647.88, (+2), 37 NTENDTIIFIR, 668.37, (+2), 26 NEAGEDSALINIK, 687.36, (+2), 66 5 Alpha-globin-D; (235) gi|63013 FLSAVSAVLAEK, 617.87, (+2), 73 DYTPEVHAAFDK, 465.26, (+3), 44 AASHQEEFGAEALTR, 539.60, (+3), 81 TYFPHFDLSPGSDQVR, 622.64, (+3), 37 Alpha 1 globin; (211) gi|52138655 MFTTYPPTK, 543.28, (+2), 50 IAGHAEEYGAETLER, 549.27, (+3), 88 VVAALIEAANHIDDIAGTLSK, 707.75, (+3), 73 6 Alpha-globin-D; (316) gi|211118 AASHQEEFGAEALTR, 539.60, (+3), 75 TYFPHFDLSPGSDQVR, 622.65, (+3), 78 DVDNLSQAMAELSNLHAYNLR, 791.75, (+3), 46 NVDNLSQAMAELSNLHAYNLR+Oxidation (M),797.07, (+3), 67 Alpha 1 globin; (251) gi|52138655 MFTTYPPTK, 543.27, (+2), 54 MFTTYPPTK+Oxidation (M), 551.28, (+2), 45 IAGHAEEYGAETLER, 549.27, (+3), 88 VVAALIEAANHIDDIAGTLSK, 707.74, (+3), 86 Heat shock 10 kDa protein 1; (79) gi|45384204 VLQATVVAVGSGAR, 664.40, (+2), 62 Beta globin; (57) gi|49169791 LLIVYPWTQR, 644.89, (+2), 33 236 I. Surowiec et al. / Meat Science 89 (2011) 233237 differences inthe concentrations of theseproteins aretoosmall tobe used reliably. This is probably because chicken muscle is predominant in both kinds of meat anddifferences inconcentrations of these proteins between HDM and MRM strongly depend on the type of MRM production. 4. Conclusions An example of high-resolution separation and identication of proteins in MRM and HDM chicken samples is presented. The results show that it is possible to separate intact high and low abundance proteins from both hand-deboned and mechanically recovered meat, which can be identied by LC-MS/MS. The methodology makes it possible to identifyproteins that couldserve as MRMbiomarkers infood products. As an example of such proteins detection of potential chicken MRM markers hemoglobin subunits and those similar to myosin- binding protein C were found. The next step will include validation of results in meat samples obtained from different producers and application of the methodology for detection of MRMin meat mixtures. Identication of proteins with different concentrations in MRM and HDM samples is also a starting point for further development that would include accurate quantication of selected peptide biomarkers in the OFF-GEL meat fractions with the application of nano-LC-MS/MS methods. Selected proteins/peptides could be also used for creating more specic antibodies to be used in ELISA assays. The advantage of the approach compared to those previously used, based on SDS-PAGE separation or capillary electrophoresis is that OFF-GEL fractionation is fast andrepeatable andfractions canbedirectly analyzedby quantitative LC-MS/MS techniques, resulting in higher selectivity, accuracy and ease of use. Acknowledgments The research presented in this article was nanced by the UK Food Standards Agency, project no. Q01102. 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