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
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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. We would like to thank Leather-
head Food International for supplies of chicken material.
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