J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te / j p ro t

JPROT-00857; No of Pages 21

Love me tender: An Omics window on the bovine meattenderness network☆

Angelo D'Alessandroa, Sara Rinalduccia, Cristina Marroccoa, Valerio Zollaa,Francesco Napolitanob, Lello Zollaa,⁎aDepartment of Ecological and Biological Sciences, University of Tuscia, Largo dell'Università, snc, 01100 Viterbo, ItalybAgricultural Research Council, via Salaria, 31, Monterotondo, Italy

A R T I C L E I N F O

☆ This article is part of a Special Issue entit⁎ Corresponding author at: Tuscia University,

E-mail address: zolla@unitus.it (L. Zolla).

1874-3919/$ – see front matter © 2012 Elseviedoi:10.1016/j.jprot.2012.02.013

Please cite this article as: D'Alessandro AJ Prot (2012), doi:10.1016/j.jprot.2012.02.

A B S T R A C T

Article history:Received 16 December 2011Accepted 13 February 2012

Meat tenderness prediction is a challenging task, especially in Maremmana, an Italianautochtonous and highly appreciated beef breed. In the present study we reported anintegrated proteomics, phosphoproteomics and metabolomics overview of meattenderness in longissimus dorsi from 15 male Maremmana individuals, through thediscrimination of tender and tough groups via standard meat tenderness indicators (WBS,MFI4 h, MFI10 days, sarcomere length) and their correlation with results from Omics analyses.Results revealed that the tender meat group was characterized by higher levels of glycolyticenzymes, which were less phosphorylated and overall more active (lactate accumulationwas higher in the tender group), than in tough counterparts. Additionally, we couldobserve a higher level of oxidative stress in the tender group.No proteomics nor phosphoproteomics result hinted at the widely accepted role of calpainsand cathepsins, except for the indication of calcium homeostasis dysregulation.Nevertheless, myofibrillar degradation was monitored and related to structural proteinfragmentations.Fragmentation of structural proteins and activities of glycolytic enzymes were inverselyrelated to their phosphorylation levels, suggesting that PTMs might add further levels ofcomplexity in the frame of meat tenderness. This article is part of a Special Issue entitled:Farm animal proteomics.

© 2012 Elsevier B.V. All rights reserved.

Keywords:Longissimus dorsiMaremmana Bos taurusMeat tendernessProteomicsPhosphorylationMetabolomics

1. Introduction

In a period of international widespread economic crisis, cattlefarming still represents a leading industrial sector, with globalpopulations estimated to be 1.3 billions of cattle (www.cattle-today.com) [1].

The increasing consumer awareness on the meat safetyand quality issues prompted the European Commission tobegin supporting from 2007 two ongoing 5-year integrated re-search projects covering beef and pork production and proces-

led: Farm animal proteomLargo dell'Università snc

r B.V. All rights reserved.

, et al, Love me tender:013

sing [2]. As far as bovine meat is concerned, the ProSafeBeefproject aims to advance safety and quality in beef productionand processing, across Europe through research and innova-tion. One of the main goals of the project is to help the beefchain to develop into a more competitive and sustainable in-dustry which takes into account consumer perceptions, atti-tudes, expectations and acceptance of existing and novelmeat production and processing systems and technologies[3]. Consumer increasing awareness on the food safety andquality issue stems from the growing acknowledgment of

ics., 01100 Viterbo, Italy. Tel.: +39 0761 357 100; fax: +39 0761 357179.

An Omics window on the bovine meat tenderness network,

2 J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

the link between food consumption on the one hand, andhealth and the food traceability issue on the other [4]. Con-sumer perception of the quality issue is largely influenced bysensory enjoyment, which in turn is characterized by threemain attributes, namely flavor, juiciness and tenderness[5,6]. As far as the latter is concerned, muscle profiling allowsevaluation of the specific beef muscle characteristics thatcontribute to tenderness, including Warner Bratzler shear-force (WBS), myofibril degradation, and insoluble collagen [7].

Indeed, variations in meat quality characteristics, includ-ing tenderness, are intimately related to the biological (espe-cially biochemical) traits and genetic variations of the liveanimals and result from centuries of livestock breeding selec-tions favoring specific quality traits, such as growth attitudeand yield [8,9]. Centuries of livestock selection and the grow-ing pressure of modern farming have contributed to the crea-tion of threats to biodiversity through the reduction ofpopulation size, which posed risks for extinction of localbreeds, among which Maremmana [10]. Owing to its peninsu-lar nature and political history, Italy has a wealth of differentbreed traditions in animal farming throughout the whole ter-ritory. In particular, a renowned region in between lower Tus-cany and higher Latium, known as Maremma, hosts a numberof autochthonous bovine breeds and populations that have al-ways been relevant for the local economic sustainability ofthe Primary sector. While some of these local breeds encoun-tered in the past a dramatic loss of economic importance, asthey had been growingly replaced by cosmopolite and moreproductive breeds, consumer awareness of the quality issuehas led to rediscover the peculiarities of regional breeds suchas Maremmana. Maremmana, named after the Maremma dis-trict, belongs to the Podolian stock and is considered to be adirect descendant of the now extinct aurochs [11,12]. It is hy-pothesized that the first introduction of the Podolian cattlebreeds in Italy followed the massive migrations of thehuman Indo-European population from Asia and north-eastEurope during the Neolithic era (9000–8000 years ago) [11].Maremmana is a very rustic cattle characterized by solidity,skeletal strength and good muscle tone, as it has beenexploited for centuries in the Italian countryside to helpworkers in the hardest task. The European Federation of AnimalScience (EAAP), in the frame of the cattle network (http://www.cattlenetwork.net/Breeds/maremmana.htm) reports that herd-book registration for Maremmana started in 1935, when thetotal breed population of Maremmana cattle was estimated at274,000 individuals. During the 1930s and '40sMaremmana cattlewere exported to Hungary and Croatia to improve the HungarianGrey and Istrian, respectively, thus resulting in a population sizeof 170,000 individuals.

Since then numbers have declined dramatically, due toland reforms and mechanization. Always according to theEAAP records, although the breed has been included in theItalian Beef Cattle Breeders Association together with theMarchigiana, Romagnola, Chianina and Apulian Podolian, bythe mid 1960s total extinction was predicted. Subsequentlythe Maremmana recovered in the period 1965–75 as it hademerged as the only breed able to adapt to the environmentalconstraints of the hilly areas of the Maremma. From 30,000animals in the mid 1960s the breed reached 60,000 in 1975.Currently, the Maremmana population consists of around

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

20,000 individuals (http://www.presidislowfood.it/ita/dettaglio.lasso?cod=19), under the protection of the Slow Food Organiza-tion which aims at preserving this highly appreciated beefbreed. However, while Maremmana meat has high nutritionalquality, on the other hand it is penalized because it is usuallyrather tough [11]. The present investigation is thus aimed todelve into the tenderness issue in Maremmana.

In the frame of meat tenderness, one of the most exten-sively and long-time investigated muscles is the longissimusdorsi [13–15], which is a valuable cut for steak (ribeye, striploinor t-bone steaks).

First approaches to longissimus dorsi tenderness envisagedbiochemical and mechanical investigations [7,13–15]. Howev-er, advancements over the last ten years in the field of prote-omics have brought about significant advancements in theunderstanding of the biochemistry behind post mortem bovinemeat tenderization [16–34], as it has been recently reviewed[1,35,36]. A role has emerged for structural proteins, glycolyticenzymes and heat shock proteins (HSPs)/chaperones in theframe of muscle tenderization after slaughter [16–34,36], al-though the biological mechanisms underpinning these hy-potheses have not been fully understood, often due to thescarce integration of proteomics results with standard meattenderness indicators collected by zootechnicians.

In this view, we recently performed a correlation analysisbetween meat quality indicators (post mortem pH, Water Hold-ing Capacity, and Minolta values) and the transcriptomics (viamicroarray) proteomics and metabolomics profiles of longissi-mus muscles from Casertana and Large White pigs [37,38].Analogously, we hereby integrated tenderness-related param-eters (WBS; collagen insolubility; myofibril degradation at 48 hand 10 days after slaughter; sarcomere length) with the re-sults obtained from proteomics (2DE and MALDI-TOF TOFidentification of differential proteins between tender andtoughmeat; Titanium dioxide enrichment and CID-ETD deter-mination of phosphorylation sites; protein–protein interac-tion modeling and gene ontology – GO – term enrichment)and results obtained through HPLC–MS metabolomics. Statis-tical analyses are then reported in order to highlight and dis-cuss significant correlations among parameters obtainedthrough standard meat tenderness indicators and Omics ap-proaches. Since meat tenderization is a multi-factorial pro-cess, we decided to reduce the number of biological variableswhich could likely affect meat tenderness (breed, muscletype, muscle area), by performing all the analyses on thesame muscle from the same individuals of the same breed.The objective of this study was thus to assess whether somebiological factors at the protein or metabolic level could bepredictor of beef tenderness in Maremmana, an increasinglyappreciated beef breed where meat tenderness is a challeng-ing issue.

2. Materials and methods

2.1. Animals

Animal handling followed the recommendations of EuropeanUnion directive 86/609/EEC and Italian law 116/92 concerninganimal care. In a commercial dairy farm located in Latium

An Omics window on the bovine meat tenderness network,

3J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

(Italy) under the supervision of Prof. Napolitano, we selected15 male Bos taurus breed Maremmana individuals (averagelive weight of 641.7±27.8 kg), which were of the same age(621.2±22.5 days) and fed the same diet.

Analyses to measure standardmeat tenderness indicators,proteomics and metabolomics analyses were performed onsamples harvested at 24 h post mortem (except where specifiedotherwise) in longissimus dorsimuscle (8th thoracic vertebra) ofMaremmana animals.

2.1.1. Standard meat tenderness indicatorsAt slaughter, carcass weight was recorded, and dressing per-centage calculated by dividing the warm carcass weight bythe shrunk live weight of the animal and expressing the resultas a percentage.

2.1.1.1. Warner Bratzler shear-force (WBS). For determina-tion of cooking loss and WBS values, samples were weighedand then cooked into a plastic bag in a water bath at 75 °Cuntil an internal temperature of 71 °C was achieved.

After cooling overnight at 2–5 °C, 6 muscle cores(1×1×3 cm) were cut parallel to the long axis of the muscle fi-bers andWBS values were taken on the cylindrical cores usingan Instron apparatus (Instron Ltd., UK) equipped with a WBSdevice, as in AMSA [39]. The texture analyzer was set with a25 kg load cell and a crosshead speed of 200 mmmin−1.

2.1.1.2. Myofibril degradation. The myofibril fragmentationindex (MFI) was determined on frozen samples taken at 2(48 h) and 10 days post mortem, as previously reported byCuller et al. [40], using a UV/VIS spectrophotometer (Ultrospec2000, Pharmacia Biotech). It was expressed as absorbance of amyofibril protein solution (concentration 0.5 mg mL−1) at540 nm multiplied by 100.

2.1.1.3. Total collagen content and collagen insolubility. Col-lagen insolubility was evaluated using Hill's method [41]. Hy-droxyproline was quantified using the spectrophotometricassay outlined by Bergman and Loxley [42], as modified byKolar [43]. A factor of 7.25 was used to convert hydroxyprolinevalues into total collagen values [44]. Collagen values arereported as mg of collagen per g of sample.

2.1.1.4. Sarcomere length. Raw meat cylinders were fixedaccording to Koolmees et al. [45]. From each cylinder, sarco-mere length of eight fiber samples for each individual was de-termined by helium neon laser diffraction (model 05-LHR-021,Melles Griot, Carlsbad, CA) as described by Cross et al. [46].

2.1.1.5. Measurement of post mortem pH. The pH of longissi-mus dorsi muscles was measured for each individual at 1 h,24 h and 48 h post mortem; about 3 g of muscle was homoge-nized in 20 ml distilled water for 15 s. The measurement wasdone using a Crison pH-meter with a combined glasselectrode.

2.2. Proteomics

2DE proteomics analyses were performed in triplicate for eachone of the 15 individuals. Image elaboration analyses were

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

then performed by comparing groups upon distinction be-tween tender (WBS<5 kg; sarcomere length>1.8 μm;myofibrildegradation10 days>90; collagen insolubility<3 mg/g) andtough (WBS>9 kg; sarcomere length<1.5 μm; myofibrildegradation10 days<70; collagen insolubility≥4 mg/g) meat, inagreement with literature [7]. On the basis of the standardmeat tenderness indicators, 7 animals were regrouped in thetender group and 7 in the tough group, while one animalwas excluded since it showed intermediate WBS (6.64 kg).

2.2.1. Sample preparationSample preparation and solubilization was performed byslight modification of the SWISS-2D PAGE sample preparationprocedure [47,48]. Frozen samples of longissimus dorsi from 15Maremmana individuals (approximately 20 mg per sample)were crushed in a mortar containing liquid nitrogen, and toremove lipids, proteins were precipitated from a desired vol-ume of each sample with a cold mix of tri-n-butyl phos-phate/acetone/methanol (1:12:1). After incubation at 4 °C for90 min, the precipitate was pelleted by centrifugation at2800×g, for 20 min at 4 °C. After removing of solution, the pel-let was air-dried and resuspended in the focusing solutioncontaining 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.8% (w/v)pH 3–10 carrier ampholyte, 40 mM Tris, 0.1 mM EDTA(pH 8.5), 2% (v/v) protease inhibitor cocktail (Sigma-Aldrich,Basle, Switzerland), and 2 mM PMSF. The protein concentra-tion of each group was determined according to Bradford [49]using BSA as a standard curve.

2.2.2. Two-dimensional electrophoresisBefore focusing, a volume of each sample containing 1 mg ofproteins was subsequently reduced (5 mM tributylphosphine,1 h) and alkylated (7.7 mM IAA, 1 h). To prevent over-alkylation, iodoacetamide (IAA) excess was neutralized byadding 10 mM dithioerythritol (DTE). IEF was performedusing eighteen centimeter IPG strips (Bio-Rad, CA, USA) pH3–10 (Bio-Rad, CA, USA) and the in gel sample rehydrationmethod. Each IPG strip (Bio-Rad, CA, USA) was rehydratedovernight with 315 μL of rehydration solution containing 7 Murea, 2 M thiourea, 4% (w/v) CHAPS, and 0.5% w/v pH 3–10 car-rier ampholyte (Bio-Rad, CA, USA). IEF was run on an ProteanIEF Cell (Bio-Rad, CA, USA) at 20 °C constant temperatureand the total product time×voltage applied was 80,000 V-h.After IEF, the IPG gel strips were incubated at room tempera-ture for 30 min in 6 M urea, 30% w/v glycerol, 2% w/v SDS,5 mM Tris–HCl, pH 8.6. The strips were sealed at the top of a1.0 mm vertical second dimensional gel (Biorad) with 0.5%agarose in 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3.SDS-PAGE was carried out on homogeneous running gels12% T 3% C. The running buffer was 25 mM Tris, 192 mMglycine, 0.1% SDS, pH 8.3 and running conditions were40 mA/gel until the bromophenol blue reached the bottomof the gel. Molecular weight marker used was Wide RangeWeight Electrophoresis Calibration Kit (Amersham Biosci-ences, UK). Gels were automatically stained with BrilliantBlue G colloidal (Sigma, St. Louis, MO, USA) following themanufacturer's instructions. Gels have been destained over-night in deionized water with blotting paper meant to gath-er Coomassie excess. Three technical replicates per samplewere performed.

An Omics window on the bovine meat tenderness network,

4 J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

2.2.3. Image analysisForty-five stained gels (3 technical replicates×15 biologicalreplicates) were digitalized using an ImageScanner and LabS-can software 3.01 (Bio-Rad Hercules, CA). The 2-DE imageanalysis was carried out and spots were detected and quanti-fied using the Progenesis SameSpots software v.2.0.2733.19819software package (Nonlinear Dynamics, New Castle UK). Eachgel was analyzed for spot detection and background subtrac-tion. Tender and tough groups were created, following the dis-criminating criteria on meat tenderness reported at thebeginning of this section. For tender versus tough group anal-ysis, only 21 gels were used per group (7 biological repli-cates×3 technical replicates for tender and tough meatyielding individuals, respectively). Within-group comparisonof protein spot numbers was determined by repeated mea-sures analysis. Among-group comparisons were determinedby ANOVA (Analysis of Variance) procedure in order to classi-fy sets of proteins that showed a statistically significant differ-ence with a confidence level of 0.05. Spots which weresignificantly different between groups (Tender versus Tough)were excised and trypsin digested for subsequent identificationby MALDI-TOF TOF. All statistical analyses were performedwith the Progenesis SameSpots software v.2.0.2733.19819 soft-warepackage [50]. After the backgroundsubtraction, spot detec-tion and match, one standard gel was obtained for each group,Tough and Tender meat. These standard gels were thenmatched to yield information about the spots of differentiallymodulated proteins. Differentially modulated protein spotswere considered significant at p-value<0.05 and the change inthe photodensity of protein spots between Tender and Toughsamples had to be more than 2 fold.

2.2.4. In-gel digestionSpots displaying statistically significantmodulation from2-DEmaps were carefully excised from the gel and subjected to in-gel trypsin digestion according to Shevchenko et al. [51] withminor modifications. The gel pieces were swollen in a diges-tion buffer containing 50 mMNH4HCO3 and 12.5 ng/mL trypsin(modified porcine trypsin, sequencing grade, Promega, Madi-son, WI) in an ice bath. After 30 min, the supernatant was re-moved and discarded; then 20 mL of 50 mM NH4HCO3 wasadded to the gel pieces, and digestion was allowed to proceedovernight at 37 °C. The supernatant containing tryptic pep-tides was dried by vacuum centrifugation.

2.2.5. Protein identification by MALDI TOF/TOFTwenty microliters of the tryptic protein digests was loadedonto activated (0.1% TFA in acetonitrile) ZipTip columns andwashed three times with 10 μL of 0.1% TFA in DD-H2O. Thepeptides were eluted with 1 μL of matrix solution (0.7 mg/mLα-cyano-4-hydroxy-trans-cinnamic acid (Fluka, Germany) in85% acetonitrile, 0.1% TFA and 1 mM NH4H2PO4) and spotteddirectly on the MALDI-TOF target plate for automatic identifi-cations (PAC384 pre-spotted anchor chip).

Proteins were identified, as previously reported [52] andper manufacturer's specifications, through an Autoflex IIMALDI-TOF/TOF mass spectrometer with the LIFT module(Bruker Daltonics) was used for mass analysis of peptide mix-tures. A peptide mixture (Peptide calibration standard I, Bru-ker Daltonics) was used for external calibration, while the

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

internal calibration was performed using the trypsin autolysisproducts. Proteins were identified by PMF using the databasesearch program MASCOT (http://www.matrixscience.com/)upon removal of background ion peaks. Accuracy was setwithin 50 ppm, while the enzyme chosen was trypsin andonly 1 missed cleavage was allowed; fixed carbamidomethyl-Cys and variable Met-oxidation, was used as optional searchcriterion. PMF-based protein identification was confirmed byMS/MS analyses of precursor ions and repeated MASCOT-based database searches. Runs were performed automaticallythrough FlexControl setting and Biotools processing of MSdata (PMF) and validation of identifications through MS/MS(LIFT analysis) on the three most intense ion peaks.

2.3. Phosphoprotein enrichment and identification ofphosphorylated peptides

2.3.1. In-solution digestionFrozen samples were crushed to powder in amortar under liq-uid nitrogen and then solubilized for 1 h at 4 °C with constantrotation in a buffer containing 7 M urea, 2 M thiourea, 4% (w/v)CHAPS, 50 mM Tris, 0.1 mM EDTA (pH 8.5), 2% (v/v) proteaseinhibitor cocktail (Sigma-Aldrich, Basle, Switzerland), 2 mMorthovanadate and 2 mM PMSF. Samples were then centri-fuged (16,000×g, 15 min, 4 °C) to remove cellular debris. Thesupernatant was transferred to a new Eppendorf tube andprotein estimation was subsequently performed using a 2Dquant kit (G.E. Healthcare, Castle Hill, NSW, Australia). 200 μgof protein was precipitated using methanol/chloroform asdescribed elsewhere [53] and resuspended in 7 M urea, 2 Mthiourea, 50 mM Tris–HCl (pH 8.8) to a final concentration of2 μg/μL. Proteins were reduced (DTT 2 mM, 30 min) and alky-lated (8 mM iodacetamide, 1 h). Finally, to prevent over-alkylation, iodoacetamide (IAA) excess was neutralized byadding 2 mM DTE. A new precipitation step was performedand sample was resuspended in 50 mM ammonium bicar-bonate containing 1 M urea and 0.1% SDS. Trypsin wasadded to a final protease:protein ratio of 1:50 (w/w) and incu-bated overnight at 37 °C.

2.3.2. Phosphopeptide enrichmentPrior to phosphopeptide enrichment, the digested sampleswere desalted using ZipTip C18 pipette tips (Millipore, Biller-ica, MA, USA) following the manufacturer's directions. Puri-fication of phosphopeptides was then performed accordingto Larsen et al. [54]. Briefly, tryptic peptides were diluted 5-fold in dihydroxybenzoic acid (DHB) buffer [350 mg/mLDHB, 80% (v/v) ACN, 2% (v/v) TFA] and applied to TiO2

beads (200 μg) pre-equilibrated in 50% ACN. The samplewas then washed once in DHB buffer, before being washedtwo times with wash buffer [80% ACN (v/v), 2% TFA (v/v)]to remove the DHB. The sample was finally eluted with25 μL of 2.5% ammonium hydroxide solution (pH≥10.5) andimmediately neutralized with 2.5 μL of formic acid. Allbuffers used ultrapure water and were made fresh on theday of experimentation.

2.3.3. LC–ESI–CID/ETD–MS/MSTo provide a larger list of phosphorylation sites, the TiO2-enriched samples were analyzed using a split-free nano-flow

An Omics window on the bovine meat tenderness network,

5J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

liquid chromatography system (EASY-nLC II, Proxeon, Oden-se, Denmark) coupled to a 3D-ion trap (model AmaZon ETD,Bruker Daltonik, Germany) equipped with an online ESInano-sprayer (the spray capillary was a fused silica capillary,0.090 mm o.d., 0.020 mm i.d.). For all experiments, a samplevolume of 15 μL was loaded by the autosampler onto a home-made 2 cm fused silica precolumn (100 μm I.D.; 375 μm O.D.;Reprosil C18-AQ, 5 μm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Sequential elution of peptides was ac-complished using a flow rate of 300 nL/min and a linear gradi-ent from Solution A (2% acetonitrile; 0.1% formic acid) to 50%of Solution B (98% acetonitrile; 0.1% formic acid) in 40 minover the precolumn in-line with a homemade 15 cm resolvingcolumn (75 μm I.D.; 375 μm O.D.; Reprosil C18-AQ, 3 μm, Dr.Maisch GmbH, Ammerbuch-Entringen, Germany). To identifyphosphorylation sites, two types of peptide fragmentationwere carried out in parallel in the mass spectrometer: (i) Colli-sion Induced Dissociation (CID); (ii) Electron Transfer Dissoci-ation (ETD). When CID was used a MS2 was automaticallyperformed on the three most intense MS ions, and MS3 wastriggered if one of the top three MS2 peaks correspondedwith neutral loss of 98.0, 49.0, 32.7m/z. For ETD experimentsthe reaction time was set to 100 ms using a reactant ICC of500000 allowing a maximum accumulation time for the reac-tant ion of 10 ms. A detailed description of the ETD setup ofthe ion trap instrument including the generation of the re-agent anion of fluoranthene was given previously [55]. The ac-quisition parameters for the instrument were as follows: drygas temperature, 220 °C; dry gas, 4.0 L/min; nebulizer gas,10 psi; electrospray voltage, 4000 V; high-voltage end-plateoffset, −200 V; capillary exit, 140 V; trap drive: 63.2; funnel 1in, 100 V out 35 V and funnel 2 in, 12 V out 10 V; ICC target,200000; maximum accumulation time, 50 ms. The samplewas measured with the “Enhanced Resolution Mode” at8100m/z per second (which allows mono isotopic resolutionup to four charge stages) polarity positive, scan range fromm/z 300 to 1500, 5 spectra averaged, and rolling average of 1.The “Smart Decomposition” was set to “auto”. Acquired ETD/CID spectra were processed in DataAnalysis 4.0, and deconvo-luted spectra were further analyzed with BioTools 3.2 soft-ware and submitted to Mascot search program (in-houseversion 2.2, Matrix Science, London, UK). The following pa-rameters were adopted for database searches: NCBInr data-base (release date 22/10/2011; 15 670 865 sequences; 5 387755 057 residues); taxonomy=mammalia; peptide mass toler-ance of ±0.3 Da; fragment mass tolerance of ±0.3 for CIDions and of ±1.3 Da for ETD ions; enzyme specificity trypsinwith 2 missed cleavages considered; fixed modifications:carbamidomethyl (C); variable modifications: oxidation (M),phosphorylation (STY). Phosphopeptide identificationswere accepted if the Mascot score was over the 95% confi-dence limit based on the “identity” score of each peptide.A delta ion score was calculated of all phosphopeptides con-taining more than one serine, threonine or tyrosine residuesby taking the difference between the two top ranking Mas-cot ion scores. Phosphorylation site assignments with adelta score >5 were automatically accepted [56]. All frag-mentation spectra with delta score ≤5 were manuallyinspected as to whether the phosphorylation sites were un-ambiguously determined or not.

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

2.4. Bioinformatic approaches

2.4.1. Protein–protein interaction analysisProtein species showing significant differential photodensi-ties between tender and tough meat samples were elaboratedto map protein–protein interactions through the widely-diffused software STRING 9.0 software [57]. Proteins identifiedexperimentally for tough and tender meat have been updatedin the software along with indications of the proteomic analy-sis and the species under investigation (B. taurus), in order toexclude false-positive protein–protein interactions and func-tional annotations derived from investigations on other spe-cies. This is a clear advantage over the Ingenuity PathwayAnalysis software, which is a powerful bioinformatic softwarethat had been designed for mining data from human sourcesand is thus biased towards the individuation of human biolog-ical pathways and/or pathologies [58].

An internal algorithm individuated proteins from the sub-mitted list in the STRING database and mapped them as graynodes. White nodes represented predicted interactors uponmatching against the internal database. Confidence intervalwas set to 0.700 (high confidence), additional white nodes to5 (as to reduce noise) and “interactors shown” value was setto 1, in order to obtain a network which was likely to be fo-cused around the input proteins.

2.4.2. Functional enrichment of GO termsDifferentially-expressed proteins between tender and toughmeat have been elaborated against FATIGO/Babelomics data-bases to get additional annotations on their functions andconsequently to establish some hypotheses concerning theirspecific role in the longissimus dorsi muscle. To this end, func-tional enrichment of gene ontologies (GOs) has been per-formed exploiting Babelomics 4.2 [58] tools such as FatiGO[58–60], in order to indirectly validate observations from pro-tein–protein interaction analyses.

FatiGO takes one list of genes and compares it againstthe rest of the genome upon conversion of the protein en-tries into a list of GO terms, using the corresponding gene-GO association table. Then a Fisher's exact test is used tocheck for significant over-representation of GO terms inthe submitted dataset against the rest of the genome. GOterms were enriched through division in three sub-categories: biological function, molecular function and sub-cellular localization [61].

2.5. Metabolomics

Metabolomic analysis has been performed as previouslyreported, with minor modifications [62,63].

Extraction and metabolite quantification method robust-ness, linearity and intra- and inter-day reproducibility havebeen confirmed, as previously reported [63].

2.5.1. Metabolite extractionSamples from each Maremmana individual were extractedfrom 150 mg of meat at 24 h from slaughtering. Sampleswere crushed in a mortar containing liquid nitrogen andextracted following the protocol by Sana et al. [64], withminor modifications as previously described [63].

An Omics window on the bovine meat tenderness network,

6 J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

Briefly, the powdered samples were resuspended by add-ing 0.15 mL of ice cold ultra-pure water (18 MΩ) to resuspendground powder; the tubes were plunged into dry ice or a circu-lating bath at −25 °C for 0.5 min and then into a water bath at37 °C for 0.5 min. To each tube was added first 0.6 mL of −20 °Cmethanol. 0.45 mL of −20 °C chloroform was added and tubeswere then mixed every 5 min for 30 min. Subsequently,0.15 mL of ice cold pH adjusted ultra-pure water (18 MΩ) wasadded to each tube and the tubes were centrifuged at 1000×gfor 1 min at 4 °C, before being transferred to −20 °C for 2–8 h.After thawing, liquid phases were recovered and an equiva-lent volume of acetonitrile was added to precipitate any resid-ual protein. The tubes were then transferred to refrigerator(4 °C) for 20 min, centrifuged at 10,000×g for 10 min at 4 °Cand the supernatants were recovered into a 2 mL tube. Col-lected supernatants were dried as to obtain visible pellets.The dried samples were re-suspended in 1 mL of water, 5%formic acid and transferred to glass autosampler vials forLC/MS analysis.

2.5.2. Rapid Resolution reversed-phase HPLCAn Ultimate 3000 Rapid Resolution HPLC system (LC Packings,DIONEX, Sunnyvale, USA) was used to perform metaboliteseparation. The system featured a binary pump and vacuumdegasser, well-plate autosampler with a six-port micro-switching valve, a thermostated column compartment. A Dio-nex Acclaim RSLC 120 C18 column 2.1 mm×150 mm, 2.2 μmwas used to separate the extracted metabolites. Acetonitrile,formic acid, and HPLC-grade water, were purchased fromSigma Aldrich (Milano, Italy).

LC parameters: injection volume, 20 μL; column tempera-ture, 30 °C; and flowrate of 0.2 mL/min. The LC solvent gradi-ent and timetable were identical during the whole period ofthe analyses. A 0–95% linear gradient of solvent A (0.1% formicacid in water) to B (0.1% formic acid in acetonitrile) wasemployed over 15 min followed by a solvent B hold of 2 min,returning to 100% A in 2 min and a 6-min post-gradient sol-vent A hold.

2.5.3. ESI mass spectrometryMetabolites were directly eluted into a High Capacity ion TrapHCTplus (Bruker-Daltonik, Bremen, Germany). Mass spectrafor metabolite extracted samples were acquired in positiveand negative ion modes, as previously described [63]. ESI cap-illary voltage was set at 3000 V (+) ion mode. The liquid nebu-lizer was set to 30 psig and the nitrogen drying gas was set to aflow rate of 9 L/min. Dry gas temperature was maintained at300 °C. Data was stored in centroid mode. Internal referenceions were used to continuously maintain mass accuracy.Data were acquired at a rate of 5 spectra/s with a storedmass range of m/z 50–1500. Data were collected using BrukerEsquire Control (v. 5.3 — build 11) data acquisition software.In MRM analysis, m/z of interest were isolated, fragmentedand monitored (either the parental and fragment ions)throughout the whole RT range. Validation of HPLC on-lineMS-eluted metabolites was performed by comparing transi-tion fingerprints, upon fragmentation and matching againstthe standardmetabolites through direct infusion with a syrin-ge pump (infusion rate 4 μL/min). Standard curve calibrationwas performed either on precursor and fragment ion signals.

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

Only the former were adopted for quantitation, as precursorion signals guaranteed higher intensity and thus improvedlimit of quantification (LOQ) and limit of detection (LOD). Tran-sitions were monitored to validate each detected metabolite.

2.5.4. Metabolite analysis and data elaborationQuantitative analyses of standard compounds were performedon MRM data against comparison to standard metabolite runs.Each standard compound wasweighted and dissolved in nano-pure water. Calibration curves were calculated as previouslyreported [63]. In brief, each standardmetabolite was run in trip-licate, at incremental dilution until LOD and LOQwere reached.The LOD for each compound was calculated as the minimumamount injected which gave a detector response higher thanthree times the signal-to-noise ratio (S/N).

Standards (equal or greater than 98% chemical purity) forglyceraldehyde phosphate (G3P), phosphoenolpyruvic acid(PEP), L-lactic acid (LA), α-ketoglutarate (KET), AMP, GMP,NAD+, NADH, glutathione (GSH), oxidized glutathione (GSSG),creatine (CREAT), phosphocreatine (PCr) and glycerol 3-phosphate were purchased from Sigma Aldrich (Milan). Stan-dards were stored either at −25 °C, 4 °C or room temperature,following manufacturer's instructions.

LC/MS data files were processed by Bruker DataAnalysis 4.0(build 234) software. Files from each run were either analyzedas .d files or exported as mzXML files, to be further elaboratedfor spectra alignment, peak picking and quantitation.

MRM data were collected for each group and graphed asfold-change variation of average quantities in CA against LWaveraged counterparts (statistically significant values=p<0.01and fold-change≥2) through the software GraphPad Prism 5.0(GraphPad Software Inc.), as previously reported [63].

2.6. Statistics

Data elaboration was performed with Excel 2007 (Microsoft,Redmond, USA) and GraphPad Prism 5.0 (GraphPad SoftwareInc). Correlations were calculated through comparing matricesof raw values for age, live weight, dressing percentage, WBS,sarcomere length,MFI values at 48 h and 10 days after slaughterand insoluble collagen concentrations for eachMaremmana in-dividual, separately. For proteomics data, 5 proteins of interestwere taken into account and treated as follows. Single ormulti-ple protein spots accounting for the sameproteinwere includedin the analysis. Spot volumes were normalized against thebackground in Progenesis SameSpots. For multiple spots, spotvolumes were summed. Then, normalized volumes of singlespots (or summed multiple spots in two cases) were averagedamong technical replicates, as to obtain one single averagedand normalized spot for each biological replicate for eachgroup (tender and tough), in agreement with our previous in-vestigation [37]. Analogously, for metabolite analyses massspectra counts were averaged among technical replicates fortwometabolites of interest (LA andGSSG) as to obtain one singlevalue for each metabolite for each individual. Data were corre-lated and plotted as in Kusec et al. [65] and D'Alessandro et al.[37], through direct analysis for the matrices of values for eachindividual for each tested couple of parameters. Absolute valuesclose to 0 indicate low correlation, while │values│≈1 indicatehigh correlation.

An Omics window on the bovine meat tenderness network,

Tab

le1–Stan

dard

mea

ttende

rnes

sindica

tors.

Marem

man

a(Bos

taurus

)Age

(day

s)W

eigh

t(kg)

Dressing

(%)

Warner

Bratzler

shea

rforce(kg)

Sarcom

ere

length(μm

)Myo

fibrillar

frag

men

tation

inde

x(48h)(MFI)

Myo

fibrillar

frag

men

tation

inde

x(10d)

(MFI)

Inso

luble

colla

gen

(mg/g)

pH (1h)

pH (24h)

pH (48h)

Ten

der(av

g±S.D.)

626.5±12

.065

6.5±4.95

58.19±1.30

2.81

±0.55

1.84

4±0.04

564

.55±4.88

109.65

±2.61

2.79

±0.36

6.87

±0.03

5.54

±0.02

5.48

±0.02

Tou

gh(avg

±S.D.)

618.5±13

.462

9.5±34

.65

55.62±0.26

10.66±1.43

1.45

3±0.06

852

.9±1.41

68.96±1.33

3.95

5±0.02

7.01

±0.01

5.58

±0.01

5.51

±0.01

7J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

3. Results and discussions

Although bovine meat tenderness is a long-time sought afterissue [66], the efforts to shed light on the mechanisms behindskeletal muscle conversion to meat and meat tenderizationare still largely debated [36,67]. Several biochemical observa-tions have been regrouped to build up a body of knowledgeabout meat tenderization yet in the late '70s [66], which ulti-mately attributed meat organoleptic properties to: (i) changesin the sarcoplasmic proteins, myofibrillar proteins (such aspartial dissociation of actomyosin, cleavage of disulfide link-ages, depolymerization of F-actin filaments, cleavage of myo-sin filaments, disorganization of Z-bands and the troponin–tropomyosin complex), sarcolemma; (ii) alterations to theconnective tissue elements (collagen fibrils, ground sub-stance); (iii) the differential effect of the post mortem forma-tion of lactic acid (H+ ion concentration) on the intra- andextracellular components of muscle; (iv) the role of lysosomalcathepsins and calcium-dependent calpains; and (v) the roleof protein degradation via proteasome [68].

More recently, a role has been suggested for apoptosis in theevents characterizing muscle conversion to meat [22,36,67]. Inrecent years, only a handful of proteomics studies have beenpublished about investigations on bovine meat tendernessthrough Omics approaches on the basis of a direct correlationof experimental outputs (from proteomics or metabolomics) tomeat quality (and in particular, tenderness-related) parameters[19,22,36].

3.1. Meat tenderness evaluation through standard meattenderness indicators

Tenderness is the organoleptic meat property which affectspalatability to the largest extent and is the driving factor ineconomic terms [36,69].

Sensory traits and classic parameters have been intimatelyrelated upon decades of research in the field of bovine meatquality, culminating in the composition of tenderness panelsfor each muscle in 2005 [7]. This body of knowledge helpedus to categorize samples derived from the longissimus dorsi ofthe 15 individuals investigated in the present study into ten-der and tough meat.

In details (Table 1), slaughtering at similar age (626.5±12.0vs. 618.5±13.4 days) resulted in comparable weight (656.5±4.95 vs. 629.5±34.65 kg) and dressing percentages (58.69 vs.55.62%) for tender and tough meat-producing individuals. Onthe other hand, tender meat was characterized by i) signifi-cantly lower WBS (2.81 vs. 10.66 kg, p<0.05); ii) higher sarco-mere length (1.844 vs. 1.453 μm); iii) higher average myofibrildegradation10 days (109.65 vs. 68.96); and iv) lower concentra-tions of insoluble collagen (2.79 vs. 3.955 mg/g).

Table 2 reports Pearson's correlation coefficients calcu-lated upon correlation of individual characteristics (age,weight, dressing percentage) and standard meat tendernessindicators (WBS, sarcomere length, MFI48 h, MFI10 d, insolu-ble collagen content, pH1h and pH24h) for each Maremmanaanimal.

Live weight demonstrated a remarkable correlation(r=0.7369) with increasing dressing percentage (Table 2).

Please cite this article as: D'Alessandro A, et al, Love me tender: An Omics window on the bovine meat tenderness network,J Prot (2012), doi:10.1016/j.jprot.2012.02.013

Table 2 – Pearson's correlation coefficients between the investigated standard meat tenderness indicators and proteomics/metabolomics parameters in Maremmana.Absolute values higher than 0.75 are highlighted in gray. Apex numbers indicate the number of the spots in Table 3 and Fig. 2 from which averaged spot volumes wereobtained through statistical Analysis via Progenesis SameSpots.

Tough Age(days)

Weight(kg)

Dressing(%)

Wb(kg)s

Sarcomerelength(μm)

48 hmyofib.degrad

10 daysmyofib.degrad

Insolublecollagen(mg/g)

pH(1 h)

pH(24 h)

TPI2383 ENO1528 PGM1533 Lactate GSSG HSP271770 HSPA83092

Age (days) 1Weight (kg) 0,0804 1Dressing (%) −0,6141 0,7369 1Wbs (kg) 0,2992 −0,3734 −0,09617 1Sarcomerelength (μm)

0,3506 0,5365 0,1877 −0,8181 1

48 h myofib.degrad

0,2705 0,0382 −0,1526 −0,4858 0,4805 1

10 days myofib.degrad

0,0020 0,2323 0,1846 −0,7933 0,6711 0,5744 1

Insolublecollagen (mg/g)

−0,2793 −0,4681 −0,0854 0,6654 −0,6304 −0,2453 −0,6618 1

pH (1 h) −0.3504 0.1507 −0.8453 0.9837 −0.9650 −0.9468 −0.9918 0.9253 1pH (24 h) −0.4528 0.7640 −0.3303 0.6836 −0.8490 −0.4832 −0.7316 0.9149 0.7107 1TPI2383 −0,8964 0,6640 −0,6103 −0,7501 0,5350 0,4251 0,6071 −0,6974 −0.6245 −0.5247 1ENO1528 −0,5037 0,2856 −0,8553 −0,9983 0,9360 0,8909 0,9793 −0,9409 −0.9837 −0.7241 0,7546 1PGM1533 −0,7186 0,5178 0,0945 0,1101 0,2156 0,2083 0,0998 −0,00813 −0.0700 −0.1318 0,7081 0,1016 1Lactate 0,7581 0,10313 −0,5203 −0,2874 0,4181 −0,6369 0,2314 −0,0905 −0.5680 −0.6423 0,8485 0,4997 0,8640 1GSSG 0,6399 −0,4448 0,2450 −0,7383 0,8881 −0,4485 0,8045 −0,5565 −0.5001 −0.6329 0,9532 0,6419 0,6827 0,6993 1HSP271770 0,4303 −0,3832 −0,4931 −0,4234 0,6692 0,6775 0,6014 −0,4717 0.5788 0.4455 0,2617 0,4276 0,8526 0,5008 0,3189 1HSPA83092 0,0106 −0,0648 −0,7933 0,7760 −0,9308 −0,8781 −0,8914 0,8087 0.8761 0.7089 0,2009 −0,7839 −0,5377 0,1212 −0,1270 −0,8926 1SOD3244 0,7147 −0,4128 0,6124 0,8780 −0,6486 −0,6602 −0,7576 −0,7393 −0.7782 −0.4711 −0,9493 −0,8701 −0,5705 −0,8567 −0,8190 0,0576 −0,3796

8JO

UR

NA

LO

FPR

OT

EO

MIC

SX

X(2012)

XX

X–X

XX

Pleasecite

this

articleas:D

'Alessan

droA,et

al,Lovem

eten

der:AnOm

icswin

dowon

thebovin

em

eatten

derness

netw

ork,JProt

(2012),doi:10.1016/j.jprot.2012.02.013

Fig. 1 – Plotting of 15 different single measurements andinterpolation lines forWarner Bratzler shear force (WBS— kg)and sarcomere length (μm) (A), WBS and myofibrillardegradation at 10 days post mortem (B), myofibrillardegradation at 10 days post mortem and insoluble collagen(mg/g) (C) in Maremmana individuals. Interpolation lines areplotted (continuous lines) and linear correlation coefficients(r) are reported in the right portion of the graph.

9J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

The effect of slaughter age and weight was negligible in re-spect to WBS (r=0.2992 — Table 2), though we should haveexpected a slightly higher age-related increase in WBS,according to Fang et al. [70].

Soon after slaughter, post mortem glycolysis gradually slowsas glycogen levels drop and acidification occurs through lac-tate and proton accumulation [71]. Lactate accumulation isthus accompanied by pH drop. Therefore, we hereby mea-sured pH at 1 h, 24 h and 48 h post mortem both in tender andtough meat groups (Table 1). In particular, the highest pH im-mediately after slaughter was measured in tough meat (7.01±0.01), while the lowest ultimate pH at 48 h after slaughter wasrecorded in tender individuals (5.48±0.02). This is suggestiveof a higher glycolytic rate in the tender meat group even be-fore slaughter, probably due to higher levels of glycolytic en-zymes in muscles from this group, which utterly translatesinto lowest pH after two days from the sacrifice of the animal(Table 1). Consistent with this hypothesis, in the presentstudy pH at 1 h after slaughter appeared to be a good predictorof meat tenderness (Table 2), as it was proportional to WBS(r=0.9837) and insoluble collagen content (r=0.9253), while itshowed a significant negative correlation with sarcomerelength (−0.9650), MFI48 h and MFI10 d (−0.9468 and −0.9918, re-spectively). Besides, pH immediately after slaughter was alsoa predictor of ultimate pH (pH24 h was used instead of pH48 h

since at two days post mortem the pH drop had already reacheda plateau and was not adequate to perform correlations),since values calculated for pH at 1 h post mortem displayedpositive correlations with those collected after 24 h(r=0.7107). Concordantly, pH24h was related to meat tender-ness as well (r=0.6836 against WBS, Table 2).

As glycolysis rate falls while ATP is rapidly hydrolyzed toreach a critically low concentration, rigor mortis occurs sincethe nucleoside triphosphate is necessary to maintain a re-laxed, uncontracted sarcomere. Therefore, the muscles startto shorten to some degree, the extent to which is dependenton pre rigor temperature [72]. When ATP levels fall below acritical threshold, the myosin heads start to become perma-nently bound to actin filaments, which result in formation ofthe actomyosin complex [73] and this causes themuscle to be-come inextensible, resulting in tougher meat. This contrac-tion results in a reduced space for water to reside betweenthe myofilaments [73]. However, sarcomere length showedpositive correlation with age, weight and dressing percentage,although results were not significant in either case.

Meat tenderization is mediated by the activation of severalpathways [36], and one of the most extensively studied eventstaking place in muscles after slaughter is proteolysis leadingto myofibrillar degradation. Although it is largely controversialwhether μ-calpains, cathepsins, proteasome through sumoyla-tion or caspases actually play the main role in myofibril degra-dation phenomena [74], it is still possible to determine theextent of proteolytic phenomena leading to myofibril degrada-tion through directmeasurements of theMFI. HigherMFI corre-sponds to most intense fragmentation events and,subsequently, to tender meat. In particular, MFI has beenshown to reach a plateau in bovine meat within 7 to 10 daysafter slaughter [75]. This is the reason why we decided to per-form the analysis at 48 h (half-plateau level [75]) and 10 days(plateau level [75]). No significant correlation was observed

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

between MFI values, either at 48 h and 10 days after slaughter,and slaughter age, weight or dressing percentage.

Intramuscular connective tissue, which is reflected by in-soluble collagen concentrations, is one of the most importantcomponents affecting meat tenderness. Insoluble collagencontents showed a moderate (r=−0.2793) correlation withage and higher, albeit not significant, with slaughter weight(r=│0.4681│ — Table 2).

Comparisons among tenderness-relatedparameters outlinedthat WBS had a negative correlation with sarcomere length (r=−0.8181, p<0.01; Fig. 1A), in agreementwith Li et al. [76]. High cor-relation between WBS and MFI was observed at plateau levels(10 days after slaughter), albeit not at 48 h (Fig. 1B). Sarcomerelength andMFI at 10 days after slaughter was positively correlat-ed among each other (r=0.6711— Table 2).

An Omics window on the bovine meat tenderness network,

Table 3 – Proteomics of Longissimus dorsi in Maremmana: Tender vs Tough Meat.

N°spot

Foldchange

Anova(p-value)

MW kDa observed/expected

pI observed/expected

N° of peptidesidentified

N° of unmatchedpeptides

Sequencecoverage

Mascotscore

NCBIaccession number

Protein ID [Bos taurus] Abbreviations

Up-regulated in TENDER839 3.2 0.0019 85.0/42.9 6.63/6.63 3 18 20% 153 gi|4838363 Creatine kinase M chain CKM

85.0/25.1 6.63/5.26 3 5 21% 133 gi|115660 Beta-casein CSN2926 3.8 0.0047 82.0/42.9 6.7/6.63 12 9 30% 105 gi|4838363 Creatine kinase M chain CKM1084 3.7 0.0033 79.0/42.9 6.63/6.63 10 15 27% 99 gi|4838363 Creatine kinase M chain CKM1086 3.8 0.0033 79.0/42.9 6.7/6.63 17 13 38% 138 gi|4838363 Creatine kinase M chain CKM1142 3.6 0.0034 78.0/42.9 6.63/6.63 14 19 38% 113 gi|4838363 Creatine kinase M chain CKM1149 3.8 0.0034 78.0/42.9 6.7/6.63 15 8 34% 148 gi|4838363 Creatine kinase M chain CKM1416 2.7 0.0044 70.0/70.2 5.4/5.55 17 4 31% 180 gi|40254806 Heat shock 70 kDa protein 1A HSPA1A1528 2.7 0.009 61.0/61.5 6.1/6.36 13 9 31% 147 gi|116004023 Phosphoglucomutase-1 PGM1

61.0/47.2 6.1/6.44 8 9 28% 128 gi|4927286 Alpha enolase ENO11530 2.3 0.0052 61.0/70.2 6.2/5.68 20 15 34% 185 gi|73853769 Heat shock 70 kDa protein 1B HSPA1B1533 2.5 0.0046 61.0/61.5 6/6.36 17 14 27% 107 gi|116004023 Phosphoglucomutase-1 PGM11770 2.6 0.0045 50.0/17.5 5.7/6.49 4 3 15% 169 gi|61553385 Heat shock 27 kDa protein 1 HSPB11837 2.3 0.0030 47.0/47.2 5.9/6.44 3 5 4% 148 gi|4927286 Alpha enolase ENO12289 3.3 0.0031 29.0/28.5 4.8/4.80 6 3 16% 187 gi|3065929 14-3-3 protein gamma YWHAG

29.0/18.7 4.8/4.68 3 6 11% 120 gi|1181841 Fast-twitch myosin light chain 1 MYL12308 3.8 0.011 29.0/29.3 7.2/7.71 8 4 36% 102 gi|77735829 Carbonic anhydrase 3 CA32383 3.2 0.0049 27.0/26.7 6.3/6.45 4 5 13 200 gi|136062 Triose-phosphate isomerase TPI2410 2.3 0.0049 26.0/22.7 5.8/5.77 15 18 61% 200 gi|71037405 Heat shock protein beta-1 HSPB1

26.0/26.9 5.8/6.45 8 7 51% 182 gi|61888856 Triosephosphate isomerase TPI2516 3.0 0.023 21.0/20.0 6.2/6.76 3 3 5% 152 gi|27805849 Alpha-crystallin B chain CRYAB2537 2.6 0.0015 21.0/42.5 5.3/7.60 8 30 16% 305 gi|149642763 ATP-sensitive inward rectifier

potassium channel 15KCNJ15

2687 2.3 0.0038 17.0/17.0 4.1/4.14 3 4 20% 130 gi|76657564 Calmodulin 2 CAM23102 3.6 0.00011 29.0/36.0 5.1/7.74 6 7 22% 180 gi|345783993 Troponin T, fast skeletal muscle TNNT3108 2.9 0.0030 15.0/14.6 5.4/6.92 8 22 45% 274 gi|6729922 Chain A, Nmr Study Of Bovine

Heart Fatty Acid Binding ProteinFABP3

3114 2.6 0.0019 29.0/29.3 7.7/7.71 7 6 32% 86 gi|77735829 Carbonic anhydrase 3 CA33120 2.5 0.00012 20.0/17.5 5.9/5.95 6 19 37% 82 gi|115496724 Heat shock protein beta-6 HSPB63130 3.9 0.006 21.0/20.0 6.3/6.76 3 4 5% 165 gi|27805849 Alpha-crystallin B chain CRYAB3132 2.6 0.0032 20.0/20.0 6.7/6.76 3 6 13% 123 gi|27805849 Alpha-crystallin B chain CRYAB3159 3.2 0.0044 29.0/68.6 5.4/7.86 7 20 16% 73 gi|329663936 SHC-transforming protein 4 SHC4

Up-regulated in TOUGH1280 2.5 0.0048 77.0/77.2 4.6/5.47 6 20 16% 75 gi|78369374 NADPH-cytochrome P450 reductase NCP12115 2.2 0.0049 35.0/36.0 7.7/7.74 11 30 38% 78 gi|47824864 Troponin T, fast skeletal muscle TNNT2146 2.4 0.0025 30.0/22.7 6.2/5.77 9 11 51% 103 gi|71037405 Heat shock protein beta-1 HSPB12171 2.7 0.0016 30.0/30.1 6.1/6.21 3 5 3% 174 gi|21039010 Troponin T slow skeletal muscle type TNNT12280 2.5 0.0017 29.0/22.7 4.1/3.97 3 3 5% 116 gi|343183383 SH3 domain binding glutamic

acid-rich proteinSH3BGRL

2303 2.9 0.0010 29.0/29.3 7.0/7.71 4 4 3% 147 gi|77735829 Carbonic anhydrase 3 CA33092 2.7 0.0032 72.0/71.2 5.3/5.37 12 18 23% 95 gi|76253709 Heat shock cognate 71 kDa protein HSPA83211 2.4 0.0037 27.0/22.7 5.3/5.77 13 16 72% 176 gi|71037405 Heat shock protein beta-1 HSPB13244 3.0 0.0040 16.0/15.5 5.7/5.86 5 5 5% 182 gi|442754 Chain A, crystal structure solution

and refinement of the semisyntheticcobalt substituted bovine erythrocyteenzyme superoxide dismutase at 2.0 Åresolution

SOD

16.0/17.0 5.7/6.90 4 4 3% 174 gi|27806939 Myoglobin MB16.0/13.9 5.7/10.42 3 3 17% 124 gi|119918149 Histone H2A.2 HTA2

10JO

UR

NA

LO

FPR

OT

EO

MIC

SX

X(2012)

XX

X–X

XX

Pleasecite

this

articleas:D

'Alessan

droA,et

al,Lovem

eten

der:AnOm

icswin

dowon

thebovin

em

eatten

derness

netw

ork,JProt

(2012),doi:10.1016/j.jprot.2012.02.013

11J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

When investigating 14 bovine muscles from Swiss Brownyoung bulls, Torrescano et al. [77] found a good relationshipbetween beef tenderness and collagen content (r=0.723) orits solubility (r=0.661). In the present study, we could observea poor correlation between WBS and total collagen content(r=0.1695), while higher correlation was observed whencomparing trends for WBS and insoluble collagen (r=0.6654).These inconsistencies with the results reported by Torrescanoand colleagues [77] might stem from breed specific peculiari-ties which characterize Swiss Brown and Maremmana maleindividuals, respectively. On the other hand, negative correla-tion was observed between insoluble collagen levels and sar-comere length (r=−0.6304) and myofibrillar degradation (r=−0.6618 – Fig. 1C – Table 2), in line with our expectations [7,76].

3.2. Proteomics and bioinformatics

Proteomics analyses on bovine meat have been extensivelyperformed over the last decade [16–36], as it has been recentlyreviewed [1,35,36]. In the present study, 2DE differential ana-lyses between those meat samples displaying tender ortough properties (as explained in the previous paragraph)resulted in the identification of 35 differential protein spots(26 up-modulated in tender meat samples, 9 in tough counter-parts), out of which 28 unique gene products were identifiedthrough MALDI TOF mass spectrometry (PMF) and validatedvia TOF TOF (in LIFT mode) (Table 3; Fig. 2).

Protein identifications are reported inTable 3, alongwith spotnumbers, fold-change variations and p-values from ANOVA test-ing, experimentally observedMWs and pIs in relation to the the-oretically expected ones and protein abbreviations.

Due to the limited number of unique gene product entries,gene ontology (GO) term enrichment for biological and molec-ular functions and cell compartment (Table 4) was performedon the overall differential dataset to increase significancy ofthe results. The cell compartment was correctly identified asmyofibril (GO:0030016), contractile fiber (GO:0043292) or actin

Fig. 2 – Two-dimensional gel electrophoresis of Maremmana tenproteins. IEF pH range is 3–10, 12% T 3% C Acrylamide. Gels havebeen elaborated with Progenesis SameSpots (Nonlinear Dynamic(3 technical replicates for 7 biological replicate samples both for

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

cytoskeleton (GO:0015629) due to the presence of proteins in-volved in the actin/tropomyosin complex of regulatory pro-teins (TNNT3, TNNT1, MYL1) or proteins indirectly related tostructural functions (CRYAB, HSPB1). As far as biological func-tions, proteins involved in responses to unfolded proteins(GO:0006986) and to organic substance (GO:001033) werefound to be significantly differentially-enriched against therest of the bovine genome, along with proteins involved inskeletal muscle contraction (GO:0003009). GO term enrich-ment for molecular functions confirmed these results, indi-cating that proteins showing differential photodensitiesmainly accounted for activities related to binding to unfoldedproteins (GO:0051082) or tropomyosin binding (GO:0005523).Further information could be grasped upon protein–proteininteraction analysis of the differential proteins through String9.0, based upon experimental evidence on Homo sapiens ortho-logues (Fig. 3). Four main clusters could be individuated,which could be summarized as follows: i) protein folding andoxidative stress-related proteins (CRYAB, HSPA1A, HSPA8,HSPB1, HSPB6, YWHAG, SOD1); ii) proteins involved in metab-olism and, in particular, glycolysis (CKM, ENO1, PGM1, TPI1);iii) structural/regulatory proteins of contractile fibers (CA3,CSN2, FABP3, MB, POR, MYL1, TNNT1, TNNT3); and iv) signal-ing protein, including kinases (CAMK2B, YWHAG, SH3BGRL,SHC4). Notably, whether the layout maybe different due to in-trinsic discrepancies among protein–protein interaction soft-wares [57], the biological read out of the network and thehigher degree nodes (protein nodes displaying the highestnumber of interactors — ENO1, HSPA1A, HSPA8, MYL) sharedan overlapping structure with the recent bioinformatic reportby Picard's group [36].

3.2.1. Muscle fibers: fiber type variability in the longissimusdorsi muscle does not justify inter-individual tendernessvariationBovine longissimus dorsimuscles were previously analyzed [78]and found to contain only 17–25% type 1 (slow twitch) fibers,

der (left side) and tough (right side) longissimus dorsi musclebeen stained with Colloidal Coomassie. Each gel image hass, NewCastle, U.K.) and represents an average of 42 gelstender and tough meat), upon background subtraction.

An Omics window on the bovine meat tenderness network,

Table 4 – Significantly (p-value<0.05) up-regulated ontologies (vs the rest of the bovine genome) in tender or toughMaremmana meat.

#Index Term #1 vs # rest of thegenome (%)

p value Pivotal proteins (UniProt names)

Biological function1 Response to unfolded protein (GO:0006986) 19.05 vs 0.5 3.668 10−6 Hspa1a, Hspa1b, HSPB1, HSPA82 Response to organic substance (GO:0010033) 33.33 vs 3.34 3.701 10−6 Hspa1a, Hspa1b, HSPB1, CRYAB, HSPA8, FABP3, SOD1,3 Skeletal muscle contraction (GO:0003009) 9.52 vs 0.06 8.969 10−5 TNNT3, TNNT1,

Molecular function1 Unfolded protein binding (GO:0051082) 19.05 vs 0.53 4.731 10−7 Hspa1a, Hspa1b, CRYAB, HSPA8,2 Tropomyosin binding (GO:0005523) 9.52 vs 0.052 5.839 10−5 TNNT3, TNNT1,

Cell compartment1 Myofibril (GO:0030016) 23.81 vs 0.47 5.008 10−8 HSPB1, TNNT3, TNNT1, MYL1, CRYAB,2 Contractile fiber (GO:0043292) 23.81 vs 0.5 6.999 10−8 HSPB1, TNNT3, TNNT1, MYL1, CRYAB,3 Actin cytoskeleton (GO:0015629) 19.05 vs 1.48 2.402 10−5 TNNT3, TNNT1, MYL1, CRYAB,

12 J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

and thus mainly contain only fast twitch glycolytic fibers.However, despite the efforts and opposed results reported byseveral research groups [79–81], proteomics has not so far pro-vided clear hints on post mortemmuscle tenderness/toughnessdiscrimination via the analysis of the percentages of specificfiber types [36]. Lee et al. [79] have recently proposed an expla-nation to this phenomenon, through suggesting that “conven-tional histochemical classifications may be involved in a high

Fig. 3 – Protein–protein interaction analysis of differentially idenMaremmanamale Bos taurus individuals. Data have been elaborainteracting protein nodes could be observed, displaying proteinsportion of the map, HSPs and proteins involved in responses toregulatory proteins were regrouped including troponin and myo

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

level of error, subjectivity and it could not clearly explain vari-ety of myofibrillar protein isoforms”.

In the present study, we could observe both fast and slowtwitch isoforms of proteins showing differential modulationin tender (fast twitch myosin light chain 1 — spot no. 2289;troponin T fast skeletal muscle — spot no. 3102) or in toughmeat (troponin T fast skeletal muscle — spot no. 2115; tropo-nin T slow skeletal muscle — spot n. 2171) (Fig. 2). No evident

tified protein spots in the Longissimus dorsi muscle ofted and graphed through String 9.0. A central cluster of highlymainly involved in glycolytic energy metabolism. In the top

stress are mapped. In the bottom portion of the map, musclesin isoforms.

An Omics window on the bovine meat tenderness network,

13J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

difference was observed as far as fiber types were concerned.This was probably due to sampling precision, technical limita-tions of the 2DE approach –which is not themost suitable oneto investigate fiber type composition – and altered protein sol-ubility, as it has been suggested by Choi et al. in porcine long-issimus dorsi muscle [81]. Protein spots showing higherphotodensity and accounting for structural proteins in toughmeat (spot no. 2115 — TNNT, for example), also displayed anapparent MW higher than tender meat counterparts (spotno. 3102— TNNT) (apparent MWs were 35 kDa and 29 kDa, re-spectively — Table 3). This observation is suggestive of minorfragmentation events taking part in the latter, in agreementwith previous studies on post mortem variation on bovine long-issimus dorsi or other longissimi muscles [18,19,21]. This resultis also consistent with Choi and Kim [80], who referred to anincreased likelihood of Z-disk protein fragmentation in mus-cles which display higher levels of glycolytic enzymes, as inthe tender group in the present study (Table 3 — see the fol-lowing paragraph). The differential fragmentation of thesame fibers in the same muscle (longissimus dorsi, in this verycase) might pinpoint at a differential micro-environmentamong individuals rather than genetic properties (the animalsinvestigated in this study were closely genetically related) orrearing characteristics (animals were reared in the same envi-ronment and fed the same diet). Post-translational modifica-tions might thus have a role in justifying these differences.

3.2.2. Metabolic enzymes: higher levels of glycolytic enzymeswere related to meat tendernessAmong the differently regulated proteins, in tender meat wecould assess an increase in glycolytic enzymes (ENO1, PGM1,TPI). After slaughter, when phosphocreatine stores areexhausted, the required energy is mainly produced throughdegradation of glycogen by glycolysis. The rate of the processdepends on the type of muscle considered but, in all cases, itpersists as long as enzymes are not inhibited by acidic pH.As explained above, a prolonged ATP generation via glycoly-sis, results in longer sarcomeres and, utterly, tender meat.The mechanism could be summarized as follows: glycolysisrate positively contributes to acidification processes (lactateand proton accumulation), although pH drop negatively af-fects glycolytic enzyme activities. The increase in hydrogenions reduces electrostatic repulsion between the myofibrillarproteins and thereby decreases the repulsion between the fil-aments, which contributes to lateral shrinkage of the musclefibers and thus sarcomere extension, which translates intotender meat [74]. Lowering of the pH also results in alterationof glycolytic enzyme solubility at post mortem [81], especially ofGAPDH and PGM1 [22], while their correlation to meat tender-ness is often isoform specific (ENO1 versus ENO3 — the latterpositively correlating with toughness, for example) and mus-cle specific (controversial results can be for example obtainedthrough the analysis of longissimus thoracis and semitendinosus)[36].

Proton homeostasis can be also modulated by carbonicanhydrase (CA), one of the fastest rate enzymes, which is in-volved in proton modulation through conversion of bicarbon-ate to water and carbon dioxide, and vice versa. Threedifferential protein spots accounting for CA3 (skeletal musclespecific isoform) were individuated, two in tender (spots no.

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

2308 and 3114) and one in tough (spots n.2303) meat samples,which displayed the same apparent MW, though differentialpI (Fig. 2) as a symptom of alternative PTMs.

ATP sensitive inward rectifier potassium channel 15 (spotno.2537) was found to be apparently fragmented (apparentMW from 2DE was considerably lower than expected —≈20 kDa against 42 kDa) (Fig. 2). Over-expression of this pro-tein is linked to high glucose consumption, anaerobiosis andanti-apoptotic effects [82]. The spot no. 3108, identified asthe fatty acid binding protein 3 (Table 3), was up-regulated intender meat samples. This protein is related both to intracel-lular transport of long-chain fatty acids and their acyl-CoA es-ters, while it also has a role in the modulation of proliferationevents and induction of apoptosis in myocardial cells and bo-vine milk [83,84].

A glycolytic enzyme, triosephosphate isomerase (TPI) wasobserved to decrease in longissimus dorsimuscles of older Mar-emmana individuals, showing a negative correlation with age(r=−0.8964). TPI has been also shown to correlate with meattenderness (WBS) in porcine muscles [85], as we could herebyconfirm (r=0.7501).

A similar correlation trend was observed for PGM, whichshowed negative correlation with age (r=−0.7186) and positivecorrelation with TPI (r=0.7081), although no significant link-age to WBS was observed. TPI also positively correlated withanother glycolytic enzyme, alpha enolase (ENO1).

In the present study, we observed that ENO1 (up-regulatedin tender meat) had a significant correlation with all thetenderness-related parameters, from WBS (r=−0.9983) to sar-comere length, MFI (both at 48 h and 10 days), pH1 h, pH24 h,and insoluble collagen levels (Table 2). The central role ofENO1 in tender meat is further evidenced by the higher pres-ence in tender samples of multiple spots accounting for crea-tine kinase M (CKM), which is responsible for the creatine/phosphocreatine shuttle. Indeed, ENO1 and CKM are knownto interact in the cytosol of skeletal muscles [86]. The levelsof CKM (spots no. 839, 926, 1084, 1086, 1142 and 1149) werehigher in the tender group, where the experimentally ob-served MW was twice as the theoretically expected one(85 kDa against 42.9 kDa) suggesting detection of dimeric spe-cies, in spite of denaturing conditions of the electrophoreticrun. Indeed, the cytosolic structure of the muscle CK enzymeis made up of a dimer of muscle type (M) subunits [86].

3.2.3. Proteomics and proteolysis: hints from oxidative stressIn the present study, proteomics analyses could not highlightany substantial differences in calpains, cathepsins, proteasomesubunits or caspases, which could be related to meat tender-ness and thus to proteolytic phenomena via canonical path-ways. However, this was largely expected due to the technicalbias of the 2DE separative technique towards high-abundanceand hydrophilic proteins, which often results in the accumula-tion of déjà vuproteins [87] or the exaltation of only thosediffer-ences between highest abundant fiber and glycolysis-relatedproteins [88]. Fragmentation events were nonetheless observed(see above), in line with our expectations.

Enzyme regulation can be reflected to some extent by thepresence of higher levels of calmodulin 2 (spot no. 2687) intender meat, where it might trigger activation of calcium-dependent proteases such as calpains or rather trigger

An Omics window on the bovine meat tenderness network,

Table 5 – Phosphorylated peptides identified by either CID (neutral loss triggered MS3) or ETD after TiO2 enrichment of whole sample lysates.

TOUGH TENDER

ETD only NL ETD only NL Aldolase AGILAANESTGSpIAK (79)Alpha-crystallin B chain

Creatine kinase M-typeLSVEALNSLTpGEFK (67)GQSpIDDMoxIPAQK (50)Glyceraldehyde 3-phosphate-dehydrogenaseGAAQNIIPASpTGAAK (78)EnolaseAAVPSGASpTGIYEALELR (61)Heat shock protein beta-1 (27 kDa)QLSpSGVSEIQQTADR (114)Heat shock protein beta-6RASpAPLPGLSAPGR (70)MyoglobinHPSDFGADAQAAMoxSpK (100)Myosin light chain 2AAAEGG(SSS)VFSMFDQTQIQEFK (92, phospho-site ?*)

AAAEGGSSpSpVFSMoxFDQTQIQEFK (135)

AEGANSpNVFSMFEQTQIQEFK (81)MyotilinS(SS)RGDVNDQDAIQEK (65, phospho-site ?*) MYOZ1 proteinGGAAGTpPGVGETGTDNQAGGEGK (73)FIYENHPDVFSpDSSMoxDR (55)LIM domain binding 3 proteinVVANSpPANADYQER (77)PGM1AIGGIILTASpHNPGGPNGDFGIK (84)Synaptopodin-2AHSpPTPSLPAGWK (46)AVSSPTpAGPAPPPPWPQPAPWSQPAFYDSSER (66)Smoothelin-like proteinSQSpFGVASASSIK (75)SLSpSSGFGAMoxTASR (62)SQSpLDHHDEASELEMoxR (76)TitinITIVTEREESpPPPAVPEIPK (71)Triosephosphate isomeraseIIYGGSpVTGATCK (60)Tropomyosin alphaAISEELDHALNDMTSpI (97)AISEELDHALNDMoxTpSI (91)Tropomyosin betaAISEELDNALNDITpSL (62)Troponin T fast skeletal muscle typeALSSMoxGANY(SS)YLAK (61, phospho-site ?*)

Aldolase AGILAANESTGSpIAK (94, CID)GILAANESTGSpIAKR (70, CID)GILAANESpTGSIAK (67, CID)Alpha-crystallin B chainAPSpWIDTGLSEMR (99, CID)Bridging integratorSPSpPPPDGSPAATPEIR (54, CID)Creatine kinase M-typeLSVEALNSLTpGEFK (86, CID)EnolaseSpGETEDTFIADLVVGLCTGQIK (67, CID)LAMQEFMILPVGADSpFK (67, CID)AAVPSGASpTGIYEALELR (66, CID)Heat shock protein beta-1 (27 kDa)QLSpSGVSEIQQTADR (98, CID)GPSpWDPFR (57, CID)Heat shock protein beta-6RASpAPLPGLSAPGR (61, CID)Histidine DecarboxylaseEMSpFPSpVNGAGDDPAHSRK (53, CID)MyoglobinHPSDFGADAQAAMoxSpK (86, ETD)GLSpDGEWQLVLNAWGK (46, CID)Myosin light chain 2AAAEGGSSSpVFSMFDQTQIQEFK (115, CID)AAAEGGSSpSpVFSMoxFDQTQIQEFK (99, CID)Myosin regulatory light chain 2, ventricular/cardiac muscle isoformAEGANSpNVFSMFEQTQIQEFK (104, CID)LIM domain binding 3 proteinVVANSpPANADYQER (113, CID)PGM1AIGGIILTASpHNPGGPNGDFGIK (100, CID)LSpGTGSAGATIR (73, ETD)Smoothelin-like proteinSQSpLDHHDEASELEMoxR (62, CID)Triosephosphate isomeraseIIYGGSpVTGATCK (82, CID)Tropomyosin alphaAISEELDHALNDMoxTpSI (80, CID)AISEELDHALNDMox(TS)I (83, phospho-site ?*, CID)AISEELDHALNDMTSpI (81, CID, 919.89 m/z)AISEELDHALNDMTpSI (82, CID, 613.59 m/z)Tropomyosin betaAISEELDNALNDITSpL (102, CID)

Aldolase AGILAANESTGSpIAK (79)Alpha-crystallin B chainAPSpWIDTGLSEMR (61)Creatine kinase M-typeLSVEALNSLTpGEFK (41)Heat shock protein beta-1 (27 kDa)QLSpSGVSEIQQTADR (114)Heat shock protein beta-6RASpAPLPGLSAPGR (82)Myosin light chain 2AAAEGGSSpSVFSMFDQTQIQEFK (90)AAAEGGSSSpVFSMoxFDQTQIQEFK (96)AAAEGGS(SS)VFSMoxFDQTQIQEFK (69, phospho-site ?*) MyoglobinHPSDFGADAQAAMSpK (83)GLSpDGEWQLVLNAWGK (66)HPSDFGADAQAAMoxSpK (85)Myosin regulatory light chain 2, ventricular/cardiac muscle isoformAEGANSpNVFSMFEQTQIQEFK (92)PGM1AIGGIILTASpHNPGGPNGDFGIK (100)LSpGTGSAGATIR (61)Smoothelin-like proteinSQSpLDHHDEASELEMoxR (60)Tropomyosin alphaAISEELDHALNDMTSpI (80, 919.90 m/z)AISEELDHALNDMTpSI (91, 613.59 m/z)AISEELDHALNDMoxTSpI (95, 619.25 m/z)Tropomyosin betaAISEELDNALNDITSpL (48)

Aldolase AGILAANESTGSpIAK (89, CID)GILAANESpTGSIAK (84, CID)Alpha-crystallin B chainAPSpWIDTGLSEMR (95)Creatine kinase M-typeLSVEALNSLTpGEFK (76, CID)GQSpIDDMoxIPAQK (45, CID)Heat shock protein beta-1 (27 kDa)QLSpSGVSEIQQTADR (85, ETD)GPSpWDPFR (59, CID)Heat shock protein beta-6RASpAPLPGLSAPGR (83, CID)MyoglobinHPSDFGADAQAAMSpK (52, ETD)GLSpDGEWQLVLNAWGK (77, CID)Myosin light chain 2AAAEGGSSSpVFSMFDQTQIQEFK (103, CID)AAAEGGSpSpSVFSMFDQTQIQEFK (89, CID)AAAEGGSpSpSVFSMoxFDQTQIQEFK (77, CID)Myosin regulatory light chain 2, ventricular/cardiac muscle isoformAEGANSpNVFSMFEQTQIQEFK (125, CID)PGM1AIGGIILTASpHNPGGPNGDFGIK (100, CID)LSpGTGSAGATIR (77, ETD)Tropomyosin alphaAISEELDHALNDMTSpI (80, CID 613.59 m/z)AISEELDHALNDMTpSI (81, CID 919.90 m/z)AISEELDHALNDMoxTpSI (91, CID)Tropomyosin betaAISEELDNALNDITSpL (105, CID)

Phosphorylation sites were determined based on the difference in Mascot scores of peptides with different possible phosphorylation sites (delta score). If the delta score was greater than 5, the top ranked phosphorylated site was considered confidently determined and reported in bold. Cases of phosphorylation ambiguity (delta score less than 5) are annoted as ?*and the potential phospho-sequence is shown in parentheses. Only phosphopeptides present in all biological replicates were taken into account and the higher Mascot score obtained is shown within brackets. Only the alpha-crystallin beta phosphopeptide has been previously detected in cow as inferred by PhosphoSite Plusdatabase (www.phosphosite.org). Unique phosphorylated peptides in tender or tough meat are underlinedand highlighted in yellow.

Myosin regulatory light chain 2, ventricular/cardiac muscle isoform

APSpWIDTGLSEMoxR(72)

AAAEGGSSpSVFSMoxFDQTQIQEFK (101)

14JO

UR

NA

LO

FPR

OT

EO

MIC

SX

X(2012)

XX

X–X

XX

Pleasecite

this

articleas:D

'Alessan

droA,et

al,Lovem

eten

der:AnOm

icswin

dowon

thebovin

em

eatten

derness

netw

ork,JProt

(2012),doi:10.1016/j.jprot.2012.02.013

15J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

downstream signaling through the activation of calcium-dependent kinases [89]. However, it seems evident enoughthat such a loose correlation might not be the unique clue toproteolysis in our investigation.

Some more robust hints seem to derive from the postula-tion of the likely role of oxidative stress in mediating tenderi-zation (other than in meat color and flavor), via reactiveoxygen species (ROS)-triggered proteolysis, as it has been al-ready suggested [36,37,74].

NADPH cytochrome P450 reductase (POR) has a ferrireduc-tase activity toward hemoglobin and iron from heme group.POR was up-regulated in tough meat (Table 3; Fig. 2) alongwith myoglobin (MB) and mapped with superoxide dismutase(SOD1) in the same cluster in the network in Fig. 3. Notablyenough, it has been suggested that a slow contractile appara-tus is associated with oxidative metabolism, so anti-oxidantenzymes (SOD1 — spot no. 3244) have a negative contributionto tenderness [36]. In line with the aforementioned postulate,oxidative stress suppression resulted in tougher meat (signif-icant negative correlation between SOD1 and MFI10days — r=−0.7576; and positive correlation between SOD1 and WBSwas observed — r=0.8780 — Table 2).

3.2.4. Chaperone proteins might contribute to tendernessthrough mediation of proteolysis and induction of apoptosisThe role of HSPs in meat tenderness has been thoroughly de-bated over the last three years [22,24,36,37]. As summarized byOuali et al. [67], HSPs might contribute to tenderness-relatedphenomena through i) the formation of a complex with activecaspases (initiators or effectors) thus hindering their function;ii) the protection of target proteins (substrates) of effector cas-pases preventing or delaying their degradation; iii) the at-tempt to re-established the initial and active structure ofproteins having undergone structural damage following ei-ther the stress itself or the initiation of apoptosis; iv) trigger-ing apoptosis when stress becomes no longer sustainable.

Among heat shock proteins, HSPB1 and CRYAB play a piv-otal role in protein protection in tender meat [36,67]. In thepresent study, chaperone proteins HSPA1A and HSPA8 wererespectively positively and negatively correlated with tender-ness (Table 3), in agreement with previous observations[24,25,31,36]. In particular, HSPA8 (spot no. 3092) was positive-ly correlated with WBS and insoluble collagen, and negativelycorrelated with sarcomere length, MFI48 h and MFI10 d. Thespecificity of each HSP in terms of protein partners in the for-mation of complexes and target proteins in terms of interac-tions could explain this difference, although furthercomplexity might be introduced by post-translational modifi-cations. PTMsmight indeedmodulate protein hydrophobicity/hydrophilicity and thus alter its interacting capability and,therefore, HSP functionality, thereby explaining why wecould observe higher photodensity of spots identified asHSPB1 both in tender (spots no. 1770, 2410) and tough meat(spots no. 2146, 3211).

In tender meat, the levels of other chaperone protein werehigher as well, including alpha crystallin B (CRYAB — spotsno. 2516, 3130, 3132 in Fig. 2). As reported by Pulford et al.[90], CRYAB levels are boosted by early post mortem oxidativestress (as we should expect in tender meat), while protein sol-ubility increases at lower ultimate pH (tender meat)

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

privileging CRYAB highlighting via electrophoretic ap-proaches. CRYAB might also contribute to improve palatabili-ty of meat through protecting proteins from heat-induceddenaturation, through complexing with beta casein [91]. It isthus worthwhile to underline that beta casein was found tobe up-regulated in tender meat as well (spot no. 839 —Table 3).

Further observations suggest that apoptosis might actual-ly have a role in meat tenderization phenomena. The protein14-3-3 gamma (YWHAG) was found to be up-regulated in ten-der meat (Table 3). YWHAG is a mediator of signal transduc-tion, among which is apoptosis activation, through bindingto MDM and thus indirect activation of p53 [92].

On the other hand, in tough meat we could identify a his-tone H2 protein (although we could not rule out the exact iso-form, while the top score protein resulted to be H2AFY). A rolefor YWHAG and H2AF variants through the mediation of apo-ptosis had been anticipated through bioinformatic modelingby Guillemin et al. [36] and hereby supported by preliminarydata.

3.3. Phosphoproteomics revealed phosphorylation of disc Zstructural proteins and glycolytic enzymes in tough meat,reducing proteolysis of the former and activity of the latter

Multiple spots accounting for the same protein were observedto display the same apparent MW, albeit different pIs(Table 3). Besides, alterations were observed in the levels ofseveral kinases or phosphorylases (SHC3, SHC4, YWHAG) orproteins triggering phosphorylation cascades through down-stream activation of kinases, such as CALM2 (see previousparagraphs). Furthermore, we had already tested that phos-phorylation might have been one of the major PTMs eventsin swine skeletal muscles [38]. Therefore, in this study we per-formed a CID/ETD analysis of tender and tough meat trypticlysates upon enrichment with TiO2. Results are reported inTable 5, along with the protein name (in alphabetical order),the indication of the enriched peptides carrying phosphoryla-tions, the indication of the phosphorylated aminoacid (eitherserine — S, threonine — T, indicated with a lower case “p”next to the aminoacid abbreviation in bold font). Differentiallyphosphorylated proteins between tender and tough groupsare highlighted in yellow.

A series of proteins were found to be phosphorylated in atleast one peptide, including aldolase, CKM, CRYAB, ENO,HSPB1, HSPB6, MB, MLC2, myosin light regulatory chain 2,ORF, PGM1, smoothelin-like protein, tropomyosin alpha andbeta. However, these proteins did not appear to be differen-tially (in qualitative terms) phosphorylated in tough and ten-der meat.

Conversely, three other groups of proteins could be indi-viduated in tough meat displaying differential phosphoryla-tions on different sites or peptides against tender meatcounterparts: i) Z-line proteins (myotilin, myozenin 1 —MYOZ1, synaptopodin-2, titin, troponin T-fast skeletal muscletype); ii) glycolytic enzymes (Glyceraldehyde 3-phosphate de-hydrogenase — GAPDH, ENO, TPI); and iii) apoptosis and sig-naling activators (bridging integrator, histidinedecarboxylase). To the best of the Authors' knowledge, thisis the first time that these peptides were observed in bovine

An Omics window on the bovine meat tenderness network,

16 J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

samples, while some of them have been already reported inhuman orthologues. A representative ETD spectrum forPGM1 is shown in Fig. 4. Detected c, z and z+1 series are dis-played, along with the peptide sequence and the determinedphosphorylation site.

Interactions between Z-disc proteins regulate musclefunctions and disruption of these interactions results inmuscle disorders. Phosphorylation (i) increases interactionsof myotilin, myozenin 1 and troponin at the Z-disc line [93];(ii) modulates actomyosin complex formation [94,95], in-creasing sarcomere cohesion and reducing accessibility toproteases: this utterly results in tougher meat. Compartmen-talization plays a role as well, through 14-3-3 binding tosynaptopodin (a myopodin homologue) resulting in its re-lease from the Z-line and nuclear import [96]. Phosphoryla-tion on the PEVK element of titin has been reported in theframe of heart stiffness [97], albeit not on the hereby ob-served peptides.

Phosphorylation of glycolytic enzymes (including GAPDH)triggered by calmodulin-dependent kinase results in 3.6 foldchange increase of activity in human orthologues [98].GAPDH phosphorylation by PKCτ/λ has also been suggestedto result in non-phosphorylating activity and target the en-zyme to pre-Golgi intermediates, enhancing its participation

Fig. 4 – ETD spectrum of the triply charged ion at 763.00m/z as dnano-HPLC–MS/MS analysis. The sequence of the identified peptiddetermined phosphorylation site is indicated as Sp. The peptide bseries (c; c+1; z; z+1) are summarized below the spectrum, in theidetected ions are shown in red. (For interpretation of the referenceversion of this article.)

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

in microtubule dynamics [99]. Its presence in tough meatseems to suggest that the latter hypothesis might be more re-liable, while apparently contradictory results stem from theincomplete knowledge about the fine tuning modulation ofGAPDH activity via phosphorylation on specific aminoacidicsites rather than others (no further information could begrasped by searching in the Phosphosite database).

Another observation underpins this statement, which islinked to the reported reduced activity of alpha enolasewhen over-phosphorylated in hypertrophied left ventricle ofspontaneously hypertensive rats [100]. Analogously, TPI activ-ity was found to be reduced upon phosphorylation duringetoposide-induced apoptosis in HeLa cells [101].

The bridging integrator (BIN) protein was found to be dif-ferentially phosphorylated in tough meat. In the frame ofskeletal muscle, BIN is involved in the radial arrangement ofsarcoplasmic strands around the central nuclei, and predom-inance and hypotrophy of type 1 fibers [102]. Phosphorylationof BIN to the peptide SPSpPPPDGSPAATPEIR on the serine 298has already been reported in human orthologues [103], al-though no specific biologic properties could be outlined.

Finally, histidine decarboxylase has been shown to bephosphorylated under oxidative stress conditions, althoughfurther details are not currently available [104].

etected after TiO2 phosphopeptide enrichment ande is reported on top of the spectrum image where the

elongs to phosphoglucomutase 1 (PGM1) protein. ETD-derivingr naturally occurring sequence in the identified peptide ands to color in this figure legend, the reader is referred to the web

An Omics window on the bovine meat tenderness network,

17J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

Finally, it is interesting to underline that phosphorylationof HSPB1 on the peptide QLSpSGVSEIQQTADR (the humanorthologue of Serine-82), as we hereby observed both in tenderand tough groups (Table 5), had been reported to activate ap-optosis via triggering dissociation of the complex with Akt[105]. Although the present phosphoproteomics approachwas not quantitative, we could suggest that phosphorylationof HSPB1 might be higher in the tender group. Indeed, we ob-served higher levels of HSPB1 in spot no. 1770 in tender meat,where the experimentally observedMWwas almost three foldhigher than theoretically expected, which might be indicativeof the presence of a small oligomer complex that preservedthe native form despite the denaturing sample preparationworkflow for the 2DE analysis (Table 3). Small HSPB1 oligo-mers (50–250 kDa) are known to derive from phosphorylationof HSPB1 and are related to mechanisms triggering apoptosis[106]. This further stresses the linkage between the meat ten-derization process and apoptosis [22,36,67].

3.4. Metabolomics analyses confirmed higher glycolysisrate in tender meat, which was accompanied by higheroxidative stress

Metabolomics analyses evidenced that in tender meat,where higher levels of glycolytic enzymes (Fig. 2, Table 3) dis-playing lower levels of phosphorylations (Table 5) werereported, glycolysis metabolic intermediates and end-

Fig. 5 – An overview of the relative quantification for metabolitestender and tough meat. In (A), bars indicate the oxidized glutathprovided for tender and tough meat, as indicated. From (B) to (D)were plotted as means±S.D. of fold-change variations of relativehigher amounts in the tender meat group; values<1, higher amoin tough group=1, upon normalization). In (B), details are provide(GLY3P) and phosphoenolpyruvate (PEP). In (C), details are providare provided for creatine (CREAT), phosphocreatine (PCREAT), AM

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

products accumulated more than in tough meat (Fig. 5A–D).In particular, phosphoenolpyruvate showed a >3.0 averagefold-change increase from tender to tough meat (Fig. 5B),along with lactate, NADH, NAD+, all substantially increasingtheir relative concentrations in the tender meat samples bya factor of 2 or higher (Fig. 5C). These results are consistentwith the significant increase of glycolytic enzymes in tendermeat and, in particular, of ENO1, the enzyme responsible forthe catalysis of the conversion of 2-phosphoglycerate (2-PG)to phosphoenolpyruvate (PEP), the ninth and penultimatestep of glycolysis. Supplementary Fig. 1 shows two spectrafor multiple reaction monitoring (MRM) analyses of lactate(89.0 [M-H]−) in tender (black line) and tough (blue line)meat.

Statistical analyses showed that lactate accumulation wascorrelated to post mortem pH (Table 2) and was proportional toanimal slaughter age and, above all, to TPI and PGM levels,while no significant correlation was observed with quantita-tive trends for ENO (Table 2).

While glyceraldehyde 3-phosphate (G3P) levels did not sig-nificantly change between tender and tough samples, glycerol3-phosphate (GLY3P — Fig. 5B), a precursor to glycerol in earlylipidogenesis via production of triacylglycerols, increased sig-nificantly in tender meat samples. This observation confirmsthat tenderness might be moderately affected by muscle mar-bling [107], that is to say intra-muscular fat deposition, andcorrelates with the result about higher levels of FABP3 ob-served in tender meat (spot no. 3108 — Table 3).

analyzed in longissimus dorsi muscles from Maremmanaione/reduced glutathione (GSSG/GSH) ratios, which are, metabolomics data were averaged for each group. Resultsabundances in tender and tough groups (values>1 indicateunts in the tough meat group; the dotted line indicates levelsd for glyceraldehyde 3-phosphate (G3P), glycerol 3-phosphateed for NAD+, NADH and lactate (LA). In (D), indicative trendsP and GMP.

An Omics window on the bovine meat tenderness network,

18 J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

Phosphocreatine levels (Fig. 5D) were moderately higher intender than in tough meat (1.1045 fold change variation, nor-malized to tough meat) while creatine was not significantlylower in the tender group (0.9561 fold change variation incomparison to tough meat). Higher post mortem phosphocrea-tine reservoirs might delay a rapid boost towards glycolysisand thus prolong enzymatic activity in the tender meatgroup, which indeed showed lower pH48h (Table 1).

In porcine muscles, which are characterized by higher gly-colytic rates, rapid pH drops after slaughter are related totougher and less juicy meat [108,109], while 45 min post mor-tem pH might not be as much indicative as ultimate pH [37].

On the other hand, in the process of bovinemuscle conver-sion to meat, a moderately accelerated rate of pH decline maybe quite beneficial to the development of tenderness, as it hasbeen suggested by the electrical stimulation models [110]. Inthis view, it is worthwhile to stress that from our results it ap-pears that pH 1 h and pH 24 h (Table 2), albeit not pH 48 h (datanot shown), were good indicators of meat tenderness.

The higher relative concentration of guanosinemonophate(GMP) in tender meat prompted us to suggest that meat dis-playing tenderness properties was also more palatable interms of taste, since GMP contributes to the formation ofumami (one of the five basic tastes together with sweet,sour, bitter, and salty) enhancing molecule formed upon theMaillard reaction of guanosine 5′-monophosphate (5′-GMP)with dihydroxyacetone and glyceraldehyde, respectively [111].

Another clear hint frommetabolomics suggested that oxida-tive stress was higher in tender meat, since GSSG/GSH ratioswere higher than in tough counterparts (Fig. 5A). Statistical an-alyses revealed that trends for GSSG had a negative correlationwith WBS (r=−0.7383) and positive correlation with sarcomerelength (r=0.881) and MFI10 days (r=0.8045), supporting the hy-pothesis behind the role of oxidative stress in meat tenderiza-tion (Table 2). Besides, GSSG was inversely related to SODlevels (r=−0.8190) and positively related to lactate accumula-tion (r=0.6993) and spot intensities of glycolytic enzymes (espe-cially TPI— r=0.9532).When oxidative stress is counteracted byanti-oxidant defenses, toughermeat shouldbe expected (higherSOD levels in tough meat — Table 3) [34,112].

4. Conclusion

The goal of the present study was to delve into the Marem-mana beef tenderness issue through a multi-faceted Omicsapproach, and to correlate results with standard meat tender-ness indicators in order to understand whether predictors ofMaremmana beef tenderness existed, either at the protein ormetabolic level. Good predictors of meat tenderness were in-dividuated as the glycolytic enzyme ENO1, the chaperone pro-tein HSPA8 and the anti-oxidant enzyme SOD at the proteinlevel, and GSSG at the metabolic level.

Additionally, we could observe that the tender meat groupwas characterized by higher levels of glycolytic enzymes,which displayed lower phosphorylation levels and overallhigher activity (as it could be deduced from higher lactate ac-cumulation in the tender meat group).

A central role was confirmed for HSPs, and for the firsttime experimentally-observed for YWHAG and H2AFY, in

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

agreement with the correlation proposed by several groups[22,36,67] between meat tenderness and apoptosis.

Phosphorylation levels of structural proteins and glycolyticenzymes were inversely related to decreased myofibrillar deg-radation/protein fragmentation and enzymatic activities, re-spectively. This prompts consideration about the likely roleplayed by specific PTMs (phosphorylations, in particular) inmodulating meat tenderness. Since the pale soft exudativemeat phenotype (PSE) has been linked to the Halothane(HAL) genotype, which codes for a protein involved in calciumhomeostasis [108,109], calcium dysregulation might mediatealterations of the post mortem meat tenderization processthrough modulation of kinase activities, which in turn triggeractivatory/inhibitory phosphorylations in specific enzymes.Furthermore, calcium-dependent phosphorylation of struc-tural proteins in tough meat might represent the missingpiece of the puzzle behind the second hypothesis about a re-duced proteolytic action of endopeptidases on myofibrilsand their co-precipitation with glycolytic enzymes (reflectingaltered solubility, compartmentalization or aggregationstate).

Supplementary materials related to this article can befound online at doi:10.1016/j.jprot.2012.02.013.

Acknowledgments

This study has been supported by the “GENZOOT” and“NUME” research programs, funded by the Italian Ministry ofAgricultural, Food and Forestry Policies (Ministero delle Poli-tiche Agricole, Alimentari e Forestali).

The Authors are grateful to Cristiana Mirasole for technicalhelp in performing proteomics analyses.

R E F E R E N C E S

[1] Bendixen E, Danielsen M, Hollung K, Gianazza E, Miller I.Farm animal proteomics—a review. J Proteomics 2011;74(3):282–93.

[2] Verbeke W, Pérez-Cueto FJ, Barcellos MD, Krystallis A,Grunert KG. European citizen and consumer attitudes andpreferences regarding beef and pork. Meat Sci 2010;84(2):284–92.

[3] Miles L, Caswell H. Advancing beef safety and quality:ProSafeBeef. Nutr Bull 2008;33:140–4.

[4] Grunert KG. Future trends and consumer lifestyles withregard to meat consumption. Meat Sci 2006;74:149–60.

[5] McIlveenH, Buchanan J. The impact of sensory factors on beefpurchase and consumption. Nutr Food Sci 2001;31(6):286–92.

[6] Grunert KG, Bredahl L, Brunsø K. Consumer perception ofmeat quality and implications for product development inthe meat sector: a review. Meat Sci 2004;66:259–72.

[7] Von Seggern DD, Calkins CR, Hohnson DD, Brickler JE,Gwartney BL. Muscle profiling: characterizing the muscles ofthe beef chuck and round. Meat Sci 2005;71:39–51.

[8] Boleman SJ, Boleman SL, Miller RK, Taylor JF, Cross HR,Wheeler TL, et al. Consumer evaluation of beef of knowncategories of tenderness. J Anim Sci 1997;75:1521–4.

[9] Bruford MW, Bradley DG, Luikart G. DNA markers reveal thecomplexity of livestock domestication. Nat Rev Genet 2003;4(11):900–10.

An Omics window on the bovine meat tenderness network,

19J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

[10] Keller LT, Waller DM. Inbreeding effects in wild populations.Trends Ecol Evol 2002;17:230–41.

[11] Gigli S, Iacurto M, Giorgetti A, Bozzi R, Poli B, Franci O, et al.Caratteristiche della qualità della carne di una razzaspecializzata da carne (Chianina) ed una rustica(Maremmana) allevate in Italia. Taurus 2000;11:87–92.

[12] Moioli B, Napolitano F, Catillo G. Genetic diversity betweenPiedmontese, Maremmana, and Podolica cattle breeds.J Hered 2004;95(3):250–6.

[13] Alsmeyer RH, Thronton JW, Hiner RL. Some dorsal-laterallocation tenderness differences in the longissimus dorsimuscle of beef and pork. J Anim Sci 1965;24:526–30.

[14] Bouton PE, Ford AL, Harris PV, Shorthose WR, Ratcliff D,Morgan JH. Influence of animal age on the tenderness ofbeef: muscle differences. Meat Sci 1978;2(4):301–11.

[15] Joseph RL, Connolly J. Measurement and prediction oftenderness in six beef muscles. Meat Sci 1979;3(1):21–9.

[16] Bjarnadóttir SG, Hollung K, Høy M, Veiseth-Kent E. Proteomechanges in the insoluble protein fraction of bovinelongissimus dorsi muscle as a result of low-voltage electricalstimulation. Meat Sci 2011;89(2):143–9.

[17] Zhang Q, Lee HG, Han JA, Kim EB, Kang SK, Yin J, et al.Differentially expressed proteins during fat accumulation inbovine skeletal muscle. Meat Sci 2010;86(3):814–20.

[18] Bjarnadóttir SG, Hollung K, Faergestad EM, Veiseth-Kent E.Proteome changes in bovine longissimus thoracis muscleduring the first 48 h postmortem: shifts in energy statusand myofibrillar stability. J Agric Food Chem 2010;58(12):7408–14.

[19] Zapata I, Zerby HN, Wick M. Functional proteomic analysispredicts beef tenderness and the tenderness differential.J Agric Food Chem 2009;57(11):4956–63.

[20] Shibata M, Matsumoto K, Oe M, Ohnishi-Kameyama M, OjimaK, Nakajima I, et al. Differential expression of the skeletalmuscle proteome in grazed cattle. J Anim Sci 2009;87(8):2700–8.

[21] Jia X, Veiseth-Kent E, Grove H, Kuziora P, Aass L, Hildrum KI,et al. Peroxiredoxin-6—a potential protein marker for meattenderness in bovine longissimus thoracis muscle. J AnimSci 2009;87(7):2391–9.

[22] Laville E, Sayd T, Morzel M, Blinet S, Chambon C, Lepetit J,et al. Proteome changes during meat aging in tough andtender beef suggest the importance of apoptosis and proteinsolubility for beef aging and tenderization. J Agric FoodChem 2009;57:10755–64.

[23] Grove H, Jørgensen BM, Jessen F, Søndergaard I, Jacobsen S,Hollung K, et al. Combination of statistical approaches foranalysis of 2-DE data gives complementary results.J Proteome Res 2008;7(12):5119–24.

[24] Morzel M, Terlouw C, Chambon C, Micol D, Picard B. Muscleproteome and meat eating qualities of Longissimus thoracisof “Blonde d'Aquitaine” young bulls: a central role of HSP27isoforms. Meat Sci 2008;78:297–304.

[25] Chaze T, Meunier B, Chambon C, Jurie C, Picard B. In vivoproteome dynamics during early bovine myogenesis.Proteomics 2008;8(20):4236–48.

[26] Jia X, Ekman M, Grove H, Faergestad EM, Aass L, Hildrum KI,et al. Proteome changes in bovine longissimus thoracismuscle during the early postmortem storage period.J Proteome Res 2007;6(7):2720–31.

[27] Jia X, Hildrum KI, Westad F, Kummen E, Aass L, Hollung K.Changes in enzymes associated with energy metabolismduring the early post mortem period in longissimus thoracisbovine muscle analyzed by proteomics. J Proteome Res2006;5(7):1763–9.

[28] Chaze T, Bouley J, Chambon C, Barboiron C, Picard B.Mapping of alkaline proteins in bovine skeletal muscle.Proteomics 2006;6(8):2571–5.

[29] Jia X, Hollung K, Therkildsen M, Hildrum KI, Bendixen E.Proteome analysis of early post mortem changes in two

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

bovine muscle types: M. longissimus dorsi and M.semitendinosis. Proteomics 2006;6(3):936–44.

[30] D'Ambrosio C, Arena S, Talamo F, Ledda L, Renzone G,Ferrara L, et al. Comparative proteomic analysis ofmammalian animal tissues and body fluids: bovineproteome database. J Chromatogr B Analyt Technol BiomedLife Sci 2005;815(1–2):157–68.

[31] Bouley J, Meunier B, Chambon C, De Smet S, Hocquette JF,Picard B. Proteomic analysis of bovine skeletal musclehypertrophy. Proteomics 2005;5(2):490–500.

[32] Bouley J, Chambon C, Picard B. Mapping of bovine skeletalmuscle proteins using two-dimensional gel electrophoresisand mass spectrometry. Proteomics 2004;4(6):1811–24.

[33] Talamo F, D'Ambrosio C, Arena S, Del Vecchio P, Ledda L,Zehender G, et al. Proteins from bovine tissues and biologicalfluids: defining a reference electrophoresis map for liver,kidney, muscle, plasma and red blood cells. Proteomics2003;3(4):440–60.

[34] Picard B, Berri C, Lefaucheur L, Molette C, Sayd T, Terlouw C.Skeletal muscle proteomics in livestock production. BriefFunct Genomics 2010;9(3):259–78.

[35] D'Alessandro A, Zolla L. We are what we eat: food safety andproteomics. J Proteome Res 2011, doi:10.1021/pr2008829.

[36] Guillemin N, Bonnet M, Jurie C, Picard B. Functional analysisof beef tenderness. J Proteomics 2011;75(2):352–65.

[37] D'Alessandro A, Marrocco C, Zolla V, D'Andrea M, Zolla L.Meat quality of the longissimus lumborum muscle ofCasertana and Large White pigs: metabolomics andproteomics intertwined. J Proteomics 2011;75(2):610–27.

[38] Murgiano L, D'Alessandro A, Egidi MG, Crisà A, Prosperini G,Timperio AM, et al. Proteomics and transcriptomicsinvestigation on longissimus muscles in Large White andCasertana pig breeds. J Proteome Res 2010;9(12):6450–66.

[39] AMSA. Research guidelines for cookery, sensory evaluation,and instrumental tenderness measurements of fresh meat.Am Meat Sci Assoc and National Live Stock and Meat Board,Chicago, IL. AOAC, 1992. Official methods of analysis15th ed.; 1995. p. 139–40. 3rd supplement.

[40] Culler RD, Parrish FC, Smith GC, Cross HR. Relationship ofmyofibril fragmentation index to certain chemical, physicaland sensory characteristics of bovine longissimus muscle.J Food Sci 1978;43:1177–80.

[41] Hill F. The solubility of intramuscular collagen in meatanimals of various ages. J Food Sci 1966;31:161–6.

[42] Bergman I, Loxley R. Two improved and simplified methodsfor spectrophotometric determination of hydroxyproline.Anal Chem 1963;35:1961–5.

[43] Kolar K. Colorimetric determination of hydroxyproline as ameasure of collagen content inmeat andmeat products: NMKLcollaborative study. J Assoc Offic Anal Chem 1990;73:54–7.

[44] Goll DE, Bray RW, Huekstra WG. Age associated changes inmuscle composition. The isolation andproperties of collagenousresidue from bovine muscle. J Food Sci 1963;28:503–9.

[45] Koolmees PA, Korteknie F, Smulders FMJ. Accuracy andutility of sarcomere length assessment by laser diffraction.Food Microstruct 1986;5:71–6.

[46] Cross HR, West RL, Dutson TR. Comparison of methods formeasuring sarcomere length in beef semitendinosus muscle.Meat Sci 1981;5:261–6.

[47] Hoogland C, Mostaguir K, Sanchez J-C, Hochstrasser D,Appel RD. SWISS-2DPAGE, ten years later. Proteomics 2004;4(8):2352–6.

[48] Czegledi L, Gulyas G, Radacsi A, Kusza S, Bekefi J, Beri B, et al.Sample Preparation and Staining Methods for Two-Dimensional Polyacrylamide Gel Electrophoresis of Proteinsfrom Animal Tissue. Animal Sci Biotechnol 2010;43(1):267–70.

[49] Bradford MM. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal Chem 1976;72:248–54.

An Omics window on the bovine meat tenderness network,

20 J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

[50] Berth M, Moser FM, Kolbe M, Bernhardt J. The state of the artin the analysis of two-dimensional gel electrophoresisimages. Appl Microbiol Biotechnol 2007;76:1223–43.

[51] Shevchenko A, Wilm M, Vorm O, Mann M. Massspectrometric sequencing of proteins from silver-stainedpolyacrylamide gels. Anal Chem 1996;68:850–8.

[52] Suckau D, Resemann A, Schuerenberg M, Hufnagel P,Franzen J, Holle A. A novel MALDI LIFT-TOF/TOF massspectrometer for proteomics. Anal Bioanal Chem 2003;376:952–65.

[53] Baker MA, SmithND, Hetherington L, PelzingM, CondinaMR,Aitken RJ. Use of titanium dioxide to find phosphopeptideand total protein changes during epididymal spermmaturation. J Proteome Res 2011;10:1004–17.

[54] Larsen MR, Thingholm TE, Jensen ON, Roepstorff P,Jorgensen TJ. Highly selective enrichment of phosphorylatedpeptides from peptide mixtures using titanium dioxidemicrocolumns. Mol Cell Proteomics 2005;4:873–86.

[55] Hartmer R, Kaplan DA, Gebhardt CR, Ledertheil T, BrekenfeldA. Multiple ion/ion reactions in the 3D ion trap: selectivereagent anion production for ETD and PTR from a singlecompound. Int J Mass Spectrom 2008;276:82–90.

[56] Wang YT, Tsai CF, Hong TC, Tsou CC, Lin PY, Pan SH, et al.An informatics-assisted label-free quantitation strategy thatdepicts phosphoproteomic profiles in lung cancer cellinvasion. J Proteome Res 2010;9:5582–97.

[57] Jensen JL, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J,et al. STRING 8—a global view on proteins and theirfunctional interactions in 630 organisms. Nucleic Acids Res2009;37:412–6.

[58] Müller T, Schrötter A, Loosse C, Helling S, Stephan C, AhrensM, et al. Sense and nonsense of pathway analysis softwarein proteomics. J Proteome Res 2011dx.doi.org/10.1021/pr200654k.

[59] Al-Shahrour F, Minguez P, Tárraga J, Medina I, Alloza E,Montaner D, et al. FatiGO +: a functional profiling tool forgenomic data. Integration of functional annotation,regulatory motifs and interaction data with microarrayexperiments. Nucleic Acids Res 2007;35:91–6.

[60] Al-Shahrour F, Díaz-Uriarte R, Dopazo J. FatiGO: a web toolfor finding significant associations of Gene Ontology termswith groups of genes. Bioinformatics 2004;20(4):578–80.

[61] Medina I, Carbonell J, Pulido L, Madeira SC, Goetz S, ConesaA, et al. Babelomics: an integrative platform for the analysisof transcriptomics, proteomics and genomic data withadvanced functional profiling. Nucleic Acids Res 2010;38:W210–3.

[62] D'Alessandro A, D'Amici GM, Timperio AM, Merendino N,Zolla L. Docosohaexanoic acid-treated PACA44 cell lines andover-activation of Krebs cycle: an integrated proteomics,metabolomics and interactomics overview. J Proteomics2011, doi:10.1016/j.jprot.2011.06.006.

[63] D'Alessandro A, Gevi F, Zolla L. A robust high resolutionreversed-phase HPLC strategy to investigate variousmetabolic species in different biological models. Mol Biosyst2011;7(4):1024–32.

[64] Sana TR, Waddell K, Fischer SM. A sample extraction andchromatographic strategy for increasing LC/MS detectioncoverage of the erythrocyte metabolome. J Chromatogr BAnalyt Technol Biomed Life Sci 2008;871(2):314–21.

[65] Kusec G, Kralik G, Petriceviac A, Gutzmirtl H, Grguric D. Meatquality indicators and their correlation in two crosses ofpigs. Agric Conspec Sci 2003;68(2):115–9.

[66] Asghar A, Yeates NT. The mechanism for the promotion oftenderness in meat during the post mortem process: areview. CRC Crit Rev Food Sci Nutr 1978;10(2):115–45.

[67] Ouali A, Herrera-Mendez CH, Coulis G, Becila S, Boudjellal A,Aubry L, et al. Revisiting the conversion of muscle into meatand the underlying mechanisms. Meat Sci 2006;74:44–58.

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

[68] Wilk S, Orlowski M. Cation-sensitive neutral endopeptidase:isolation and specificity of the bovine pituitary enzyme.J Neurochem 1980;35(5):1172–82.

[69] Belew JB, Brooks JC, McKenna DR, Savell JW.Warner–Bratzlershear evaluations of 40 bovine muscles. Meat Sci 2003;64(4):507–12.

[70] Fang SH, Nishimura T, Takahashi K. Relationship betweendevelopment of intramuscular connective tissue andtoughness of pork during growth of pigs. J Anim Sci 1999;77:120–30.

[71] [71]Lomiwes D. Rapid on-line Glycogen measurement andprediction of ultimate pH in slaughter beef. PhD Thesis 2008;available at http://hdl.handle.net/10292/970; Last accessedon January 26th, 2012.

[72] Locker RH, Hagyard CJ. A cold shortening effect in beefmuscles. J Sci Food Agr 1963;14:787–93.

[73] Lawrie RA. Meat Science. Cambridge: Woodhead PublishingLimited; 1998.

[74] Pearce KL, Rosenvold K, Andersen HJ, Hopkins DL. Waterdistribution and mobility in meat during the conversion ofmuscle to meat and ageing and the impacts on fresh meatquality attributes—a review. Meat Sci 2011;89(2):111–24.

[75] Ilian MA, Ael-D B, Bickerstaffe R. The relationship betweenmeat tenderization, myofibril fragmentation and autolysisof calpain 3 during post mortem aging. Meat Sci 2004;66(2):387–97.

[76] Li CB, Zhou GH, Xu XL. Dynaimcal changes of beefintramuscular connective tissue and muscle fiber duringheating and their effects on beef shear force. FoodBioprocess Technol 2010;3:521–7.

[77] Torrescano G, Sánchez EA, Giménez B, Roncalés P, BeltránJA. Shear values of raw samples of 14 bovine muscles andtheir relation to muscle collagen characteristics. Meat Sci2003;64:85–91.

[78] Vestergaard M, Oksbjerg N, Henckel P. Influence of feedingintensity, grazing and finishing feeding on muscle fibrecharacteristics and meat colour of semitendinosus,longissimus dorsi and supraspinatus muscles of youngbulls. Meat Sci 2000;54(2):177–85.

[79] Lee SH, Joo ST, Ryu YC. Skeletal muscle fiber type andmyofibrillar proteins in relation to meat quality. Meat Sci2010;86(1):166–70.

[80] Choi YM, Kim BC. Muscle fiber characteristics, myofibrillarprotein isoforms, and meat quality. Livest Sci 2009;122:105–18.

[81] Choi YM, Lee SH, Choe JH, Rhee MS, Lee SK, Joo ST, et al.Protein solubility is related to myosin isoforms, muscle fibertypes, meat quality traits, and postmortem protein changesin porcine longissimus dorsi muscle. Livest Sci 2010;127:183–91.

[82] Akao M, Ohler A, O'Rourke B, Marbán E. MitochondrialATP-sensitive potassium channels inhibit apoptosisinduced by oxidative stress in cardiac cells. Circ Res 2001;88(12):1267–75.

[83] Zhu C, Hu DL, Liu YQ, Zhang QJ, Chen FK, Kong XQ, et al.Fabp3 inhibits proliferation and promotes apoptosis ofembryonic myocardial cells. Cell Biochem Biophys 2011;60(3):259–66.

[84] D'Alessandro A, Zolla L, Scaloni A. The bovine milkproteome: cherishing, nourishing and fostering molecularcomplexity. An interactomics and functional overview. MolBiosyst 2011;7(3):579–97.

[85] Hwang IH, Park BY, Kim JH, Cho SH, Lee JM. Assessment ofpostmortem proteolysis by gel-based proteome analysis andits relationship to meat quality traits in pig longissimus.Meat Sci 2005;69(1):79–91.

[86] Foucault G, Vacher M, Cribier S, Arrio-DupontM. Interactionsbetween beta-enolase and creatine kinase in the cytosol ofskeletal muscle cells. Biochem J 2000;346(1):127–31.

An Omics window on the bovine meat tenderness network,

21J O U R N A L O F P R O T E O M I C S X X ( 2 0 1 2 ) X X X – X X X

[87] Petrak J, Ivanek R, Toman O, Cmejla R, Cmejlova J, Vyoral D,et al. Déjà vu in proteomics. A hit parade of repeatedlyidentified differentially expressed proteins. Proteomics2008;8(9):1744–9.

[88] Ohlendieck K. Proteomics of skeletal muscle glycolysis.Biochim Biophys Acta 2010;1804(11):2089–101.

[89] Sentandreu MA, Coulis G, Ouali A. Role of muscleendopeptidases and their inhibitors in meat tenderness.Trends Food Sci Tech 2002;13:400–42.

[90] Pulford DJ, Fraga Vazquez S, Frost DF, Fraser-Smith E, DobbieP, Rosenvold K. The intracellular distribution of small heatshock proteins in post mortem beef is determined byultimate pH. Meat Sci 2008;79(4):623–30.

[91] Pulford DJ, Frost DF, Lomiwes DD, Farouk MM. Preliminarystudies to determine the chaperoning properties of bovinecasein and crystallin proteins at reducing beef muscleprotein aggregation during heating. Int J Food Sci Tech2008;43:2143–50.

[92] Jin Y, Dai MS, Lu SZ, Xu Y, Luo Z, Zhao Y, et al. 14-3-3gammabinds to MDMX that is phosphorylated by UV-activatedChk1, resulting in p53 activation. EMBO J 2006;25(6):1207–18.

[93] von Nandelstadh P, Ismail M, Gardin C, Suila H, Zara I,Belgrano A, et al. A class III PDZ binding motif in themyotilin and FATZ families binds enigma family proteins: acommon link for Z-disc myopathies. Mol Cell Biol 2009;29(3):822–34.

[94] Mazzei GJ, Kuo JF. Phosphorylation of skeletal-muscle troponinI and troponin by phospholipid-sensitive Ca2+−dependentprotein kinase and its inhibition by troponin C andtropomyosin. Biochem J 1984;218(2):361–9.

[95] Perry SV, Cole HA. Phosphorylation of troponin and theeffects of interactions between the components of thecomplex. Biochem J 1974;141(3):733–43.

[96] Faul C, Dhume A, Schecter AD, Mundel P. Protein kinase A,Ca2+/calmodulin-dependent kinase II, and calcineurinregulate the intracellular trafficking of myopodin betweenthe Z-disc and the nucleus of cardiac myocytes. Mol Cell Biol2007;27(23):8215–27.

[97] Hidalgo C, Hudson B, Bogomolovas J, Zhu Y, Anderson B,Greaser M, et al. PKC phosphorylation of titin's PEVKelement: a novel and conserved pathway for modulatingmyocardial stiffness. Circ Res 2009;105(7):631–8.

[98] Singh P, Salih M, Leddy JJ, Tuana BS. The muscle-specificcalmodulin-dependent protein kinase assembles with theglycolytic enzyme complex at the sarcoplasmic reticulumand modulates the activity of glyceraldehyde-3-phosphatedehydrogenase in a Ca2+/calmodulin-dependent manner.J Biol Chem 2004;279(34):35176–82.

[99] Tisdale EJ, Kelly C, Artalejo CR. Glyceraldehyde-3-phosphatedehydrogenase interacts with Rab2 and plays an essentialrole in endoplasmic reticulum to Golgi transport exclusive ofits glycolytic activity. J Biol Chem 2004;279:54046–52.

Please cite this article as: D'Alessandro A, et al, Love me tender:J Prot (2012), doi:10.1016/j.jprot.2012.02.013

[100] Jin X, Wang LS, Xia L, Zheng Y, Meng C, Yu Y, et al.Hyper-phosphorylation of alpha-enolase in hypertrophiedleft ventricle of spontaneously hypertensive rat. BiochemBiophys Res Commun 2008;371(4):804–9.

[101] Lee WH, Choi JS, Byun MR, Koo KT, Shin S, Lee SK, et al.Functional inactivation of triosephosphate isomerasethrough phosphorylation during etoposide-inducedapoptosis in HeLa cells: potential role of Cdk2. Toxicology2010;278(2):224–8.

[102] Nicot AS, Toussaint A, Tosch V, Kretz C,Wallgren-PetterssonC, Iwarsson E, et al. Mutations in amphiphysin 2 (BIN1)disrupt interaction with dynamin 2 and cause autosomalrecessive centronuclear myopathy. Nat Genet 2007;39(9):1134–9.

[103] Rigbolt KT, Prokhorova TA, Akimov V, Henningsen J,Johansen PT, Kratchmarova I, et al. System-wide temporalcharacterization of the proteome and phosphoproteome ofhuman embryonic stem cell differentiation. Sci Signal2011;4:3.

[104] Höcker M, Rosenberg I, Xavier R, Henihan RJ, WiedenmannB, Rosewicz S, et al. Oxidative stress activates the humanhistidine decarboxylase promoter in AGS gastric cancercells. J Biol Chem 1998;273(36):23046–54.

[105] Rane MJ, Pan Y, Singh S, Powell DW,Wu R, Cummins T, et al.Heat shock protein 27 controls apoptosis by regulating Aktactivation. J Biol Chem 2003;278(30):27828–35.

[106] Rogalla T, Ehrnsperger M, Preville X, Kotlyarov A, Lutsch G,Ducasse C, et al. Regulation of Hsp27 oligomerization,chaperone function, and protective activity against oxidativestress/tumor necrosis factor alpha by phosphorylation. J BiolChem 1999;274(27):18947–56.

[107] Wheeler TL, Cundiff LV, Koch RM. Effect of marbling degreeon beef palatability in Bos taurus and Bos indicus cattle.J Anim Sci 1994;72(12):3145–51.

[108] Gil M, Hortos M, Sarraga C. Calpain and cathepsin activities,and protein extractability during ageing of longissimusporcine muscle from normal and PSE meat. Food Chem1998;63:385–90.

[109] Laville E, Sayd T, Terlouw C, Blinet S, Pinguet J, Fillaut M,et al. Differences in pig muscle proteome according to HALgenotype: implications for meat quality defects. J Agric FoodChem 2009;57:4913–23.

[110] Takahashi G, Lochnert JV,Marsh BB. Effects of low-frequencyelectrical stimulation on beef tenderness. Meat Sci 1984;11(3):207–25.

[111] Festring D, Hofmann T. Systematic studies on the chemicalstructure and umami enhancing activity of Maillard-modifiedguanosine 5′-monophosphates. J Agric Food Chem 2011;59(2):665–76.

[112] Huff Lonergan E, Zhang W, Lonergan SM. Biochemistry ofpostmortem muscle — lessons on mechanisms of meattenderization. Meat Sci 2010;86(1):184–95.

An Omics window on the bovine meat tenderness network,