Differential abundance of proteome associated with intramuscular variation of meat quality in porcine longissimus thoracis et lumborum muscle

Accepted Manuscript Differential abundance of proteome associated with intramuscular variation of meat quality in porcine longissimus thoracis et lumborum muscle

Gap-Don Kim, Jin-Yeon Jeong, Han-Sul Yang, Sun Jin Hur PII: DOI: Reference:

S0309-1740(18)30389-9 https://doi.org/10.1016/j.meatsci.2018.11.012 MESC 7724

To appear in:

Meat Science

Received date: Revised date: Accepted date:

9 April 2018 4 October 2018 14 November 2018

Please cite this article as: Gap-Don Kim, Jin-Yeon Jeong, Han-Sul Yang, Sun Jin Hur , Differential abundance of proteome associated with intramuscular variation of meat quality in porcine longissimus thoracis et lumborum muscle. Mesc (2018), https://doi.org/ 10.1016/j.meatsci.2018.11.012

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Differential abundance of proteome associated with intramuscular variation of meat quality in porcine longissimus thoracis et lumborum muscle

Gap-Don Kima,b, Jin-Yeon Jeongc, Han-Sul Yangc,d, Sun Jin Hure ,* Graduate School of International Agricultural Technology, Seoul National University,

IP

T

a

b

CR

Pyeongchang 25354, Republic of Korea

Institutes of Green-Bio Science and Technology, Seoul National University, Pyeongchang

c

US

25354, Republic of Korea

Division of Applied Life Science (BK21 plus), Gyeongsang National University, Jinju 52828,

Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic

M

d

AN

Republic of Korea

of Korea

Department of Animal Science and Technology, Chung-Ang University, Anseong 17546,

ED

e

AC

CE

PT

Republic of Korea

*

Corresponding author: S.J. Hur, Tel. +82-31-670-4673, Fax. +82-31-675-3108, E-mail:

[email protected]

ACCEPTED MANUSCRIPT

Abstract Intramuscular variation of meat quality in the porcine longissimus thoracis et lumborum (LTL) muscle was evaluated by assessing the differential abundance of proteome components.

T

Twenty LTL muscles were individually divided into three regions (anterior, medial, and

IP

posterior) according to meat color. CIE L* and b* were higher (P < 0.05), but Warner–Bratzler

CR

shear force (WBSF) was lower (P < 0.05) in the anterior region, where myosin-1 and -2, TPM2, MLC1f, MLC2, Hsp27, and TPI1 were highly (P < 0.05) abundant. However, CIE a* , drip loss,

US

and WBSF were higher (P < 0.05) in the medial region. Glycolysis enzymes including enolase 3,

AN

ALDOA, LDHA, PGM1, and TPI1 were highly abundant in the medial and posterior region, whereas GAPDH and myoglobin were overexpressed in the medial region (P < 0.05). Therefore,

M

intramuscular variations in color, water-holding capacity, and tenderness were associated with

CE

PT

ED

differential abundance of the proteome, especially contractile, glycolysis enzymes, and Hsp27.

AC

Keywords: Intramuscular variation; Proteome; Longissimus thoracis et lumborum; Pork quality

ACCEPTED MANUSCRIPT

1. Introduction Each skeletal muscle of the animal body is unique in terms of its structure and function (Maclntosh, Gardiner, & McComas, 2006). In other words, each individual muscle has its own

T

physiological, morphological, biological, and biochemical characteristics (Konhilas, Irving, & de

IP

Tombe, 2002; McDonald, Wolff, & Moss, 1997). During conversion from muscle to meat, those

CR

characteristics strongly influence the meat quality and thus cause differences in quality among the cuts (muscles). Many studies have addressed this issue previously (Chang et al., 2003; Eggert,

US

Depreux, Schinckel, Grant, & Gerrard, 2002; Karlsson, Klont, & Fernandez, 1999). Therefore,

AN

single muscle is generally considered a basic unit in the research area of livestock and meat science. However, some muscles, such as semimembranosus (SM) and semitendinosus (ST),

M

have intramuscular variations in biological and biochemical characteristics as well as meat

ED

quality (Kim, Yang, & Jeong, 2018; Lefaucheur, Edom, Ecolan, & Butler-Browne, 1995; Nair et al., 2016). Specifically, both SM and ST exhibit two distinguishable color intensities, which are

PT

caused by the different composition of muscle fiber type and proteome (Kim et al., 2018). This

CE

issue has been mainly discussed regarding porcine and bovine SM and ST muscles to date.

AC

Longissimus thoracis et lumborum (LTL) muscle, which is the longest muscle found in the animal body, has been evaluated for its biological and biochemical characteristics in previous studies: proteome in relation to meat quality (Kwasiborski, Sayd, Chambon, Santé-Lhoutellier, Rocha, & Terlouw, 2008; te Pas et al., 2013; van de Wiel & Zhang, 2007), protein oxidation (Promeyrat, Sayd, Laville, Chambon, Lebret, & Gatellier, 2011), and proteolysis (Bee, Anderson, Lonergan, & Huff-Lonergan, 2007; Park, Kim, Lee, & Hwang, 2007). The location of LTL muscle on vertebrae or ribs should be considered because of its length and intramuscular

ACCEPTED MANUSCRIPT

variation. LTL muscle is generally distinguished as two parts: longissimus thoracis (LT) and longissimus lumborum (LL). Although each part of LTL muscle is well established in terms of biological and biochemical properties (LT: Kim, Ryu, Jeong, Yang, & Joo, 2013; Park et al., 2007; te Pas, Jansen, Broekman, Reimert, & Heuven, 2009; LL: Kwasiborski et al., 2008; te Pas

IP

T

et al., 2013), intramuscular variations of LTL muscle have not been fully assessed. Therefore, the

CR

objective of the present study was to identify and quantify the differentially abundant proteome

AN

US

in different regions of porcine LTL muscle to elucidate intramuscular meat quality variation.

M

2. Materials and methods

ED

2.1. Sample preparation and experimental design

PT

Twenty pig carcasses (10 gilts and 10 barrows, 108.4±1.5 kg) at 24 h postmortem were randomly selected at a commercial slaughter house that were slaughtered following the

CE

Livestock Products Sanitary Control Act, Republic of Korea. Whole loin (LTL muscle) between

AC

the 4th thoracic vertebrae and the last lumbar vertebrae was taken from the left side of each carcass. Each loin was cut into 15 chops with 3.3-cm thickness in consideration of loin length (51.0 to 54.5 cm) (Fig. 1). Both residues on the edge of the loin were removed. Chop numbers from 1 (anterior) to 15 (posterior) were designated serially. All chops were exposed to air for 20 min for myoglobin oxygenation, and then surface color was measured. Data for the Commission Internationale de l’Eclairage (CIE, 1978) L* (lightness), a* (redness), and b* (yellowness) were subjected to Cluster analysis (PROC CLUSTER; SAS, SAS Institute, Cary, NC, USA) with the

ACCEPTED MANUSCRIPT

average linkage method. The result was presented by PROC TREE HORIZONTAL (spaces = 2). Chop sequences # 9 and 10 were distinguished from # 11, 12, and 13 with 0.9 of average distance, and those sequences were distinguished from # 1, 3, and 5 with 1.24 of average distance. Therefore, chops were serially grouped with five chops per group, and three groups

IP

T

were classified: Anterior, # 1–5; Medial, # 6–10; Posterior, # 11–15. Five chops of each group

CR

were randomly allocated for measurements: two chops for pH and proximate composition (moisture, crude fat, and crude protein) and one chop each for drip loss, cooking loss with

AN

2.2. Protein extraction and LC-MS/MS analysis

US

tenderness, and immunohistochemistry and LC-MS/MS analysis.

Five grams of sample was obtained from each chop, and muscle proteins were extracted

M

using a lysis buffer (8 M urea and 0.1 M DTT). Muscle samples were put into a 50-mL

ED

centrifuge tube, and 25 mL lysis buffer was added. Samples were homogenized using a

PT

homogenizer (T25basic, IKA, Rawang, Malaysia) and subsequently sonicated on ice. Protein concentration was determined with the Bradford (1976) method and adjusted to 2 mg/mL (0.35

CE

mL in total). In-solution digestion was conducted using sequence-grade modified trypsin

AC

(enzyme to substrate ratio of 1:50; Promega, Madison, WI, USA) at 37°C for 24 h. The tryptic digest was desalted with Zip-Tip C18 with standard bed format (Millipore Corp., Bedford, MA, USA) and then analyzed in an Easy-nLC (Thermo Fisher, San Jose, CA, USA) and a LTQOrbitrap XL mass spectrometer (Thermo Fisher, San Jose, CA, USA) equipped with a nanoelectrospray source as described by Kim et al. (2018). In brief, the tryptic digest was separated on a C18 nano bore column (150 mm × 0.1 mm, pore size of 3 μm; Agilent Technologies, Santa Clara, CA, USA). Mobile phase A for LC separation consisted of 0.1% formic acid and 0.3%

ACCEPTED MANUSCRIPT

acetonitrile in deionized water. Mobile phase B consisted of 0.1% formic acid in acetonitrile. Chromatography was conducted with a linear gradient (5%–40% B for 40 min, 40%–65% B for 4 min, 95% B for 4 min, and 5% B for 6 min). The flow rate was maintained at 1500 nL/min. Mass spectra were obtained using data-dependent acquisition with a full mass scan (350–1200

IP

T

m/z) followed by 10 MS/MS scans. For MS full scans, orbitrap resolution was set at 15,000 with

CR

automatic gain control (AGC) at 2×105 . The AGC for MS/MS in the LTQ-Orbitrap XL mass

2.3. Proteome quantification and bioinformatics

US

spectrometer was set at 1×104 .

AN

Label-free quantification (LFQ) was conducted to identify and quantify meat proteins using the MaxQuant software (version 1.5.5.1; http://www.coxdocs.org; Cox & Mann, 2008).

M

Raw mass spectrometric data obtained from LC-MS/MS analysis were subjected to MaxQuant

ED

analysis with the following conditions: variable modifications, methionine oxidation and N-

PT

terminal acetylation; fixed modification, carbamidomethylation of cysteine; mass deviation, 20 ppm; maximum missed cleavages, 2; maximum peptide modifications, 5; and peptides, resulting

CE

from trypsin digest. Quantification was performed using the LFQ algorithm with peak

AC

normalization. Razor and unique peptides were used for LFQ and statistical evaluation. For identification of peptides and proteins, a database was derived from UniProt (release March 2018, Sus scrofa 9823, 48,896 sequences). To evaluate the protein function and protein–protein interaction, String 10.5 database (http://string-db.org), which is a search tool for the retrieval of interacting genes and proteins, was employed. The differentially expressed proteins defined by LFQ were subjected to String analysis. Pathway analysis was performed using the Gene Ontology categories including biological process, molecular function, and cellular component.

ACCEPTED MANUSCRIPT

2.4. Immunohistochemistry For evaluation of muscle biological characteristics, muscle fiber composition and crosssectional area (CSA) per fiber type were evaluated using immunohistochemistry as described by

T

Kim et al. (2013). In brief, four transverse serial sections (10 μm in thickness) were obtained

IP

using a cryostat microtome (HM525, Microm GmbH, Walldorf, Germany) at –27 °C. These

CR

sections were blocked with goat serum for 1 h at room temperature (RT). Subsequently, the sections were incubated with four primary antibodies (BA-D5, SC-71, BF-35, and 10F5; DSHB,

US

Iowa City, IA, USA) for 1 h at RT. Secondary antibodies such as biotinylated IgG and IgM were

AN

applied to the sections for 30 min at RT. Immune-peroxidase staining was conducted using an avidin–biotin complex (Thermo Fisher Scientific Inc., Waltham, MA, USA). The muscle fibers

M

were visualized using diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corp., St. Louis, MO,

ED

USA). Muscle fibers were classified into four types according to the specificity of primary antibodies to myosin heavy chain (MHC) isoforms: type I, fibers detected with BA-D5 (anti-

PT

MHC I) and BF-35 (anti-MHC I and 2a); type IIA, fibers detected with SC-71 (anti-MHC 2a and

CE

2x) and BF-35; type IIX, fibers detected with SC-71; type IIB, fibers detected with 10F5 (antiMHC 2b). Approximately 600 fibers per sample were analyzed using Image-Pro® plus 5.1

AC

program (Media Cybernetics Inc., Rockville, MD, USA). CSA, relative area composition (%), and fiber density were evaluated. The relative area composition was considered the ratio of the total CSA of each fiber type to the total measured fiber area. The fiber density (number/mm 2 ) was presented as the number of individual fibers of a given type per 1 mm2 of the fiber CSA. 2.5. Meat quality

ACCEPTED MANUSCRIPT

To evaluate the meat quality, pH, proximate composition, meat color, water-holding capacity, and tenderness were investigated. Meat homogenate (3.0 g in 27 mL of deionized water) was prepared, and the pH was measured using a pH meter (MP230, Mettler Toledo, Greifensee, Switzerland). The meat color of pork chops was measured using the Minolta Chromameter (CR-

IP

T

300, Minolta Co., Tokyo, Japan) with a D65 light source after calibration using a ceramic plate

CR

(Y = 93.5, x = 0.3132, y = 0.3198). Instrumental color values were presented as the CIE L* , a* , and b* . Moisture and crude protein contents (%) were determined using an AOAC (1995)

US

method. Crude fat was analyzed by the method of Folch, Lees, and Sloane-Stanley (1957) with modification. In brief, 5 g of the sample was homogenized with 30 mL of extraction solution

AN

(chloroform:methanol, 2:1, v/v) and then filtered with Whatman No.1 paper. The filtrate was

M

separated with 0.88% NaCl. The upper layer was removed, and the lower layer was evaporated

ED

by nitrogen gas. The remaining crude fat was presented as a percentage of initial weight. The pH, instrumental color, and proximate composition were measured in triplicate for each sample, and

PT

the average was recorded. To determine water-holding capacity, drip loss and cooking loss were evaluated. Drip loss was determined using the method of Honikel (1987) with modification.

CE

Briefly, each chop was weighed initially and suspended in a plastic zipper bag at 4 °C for 24 h.

AC

The chop was removed from the bag and weighed. Drip loss was presented as a percentage of initial weight. To measure cooking loss, each chop was packed with a plastic bag and cooked in a water bath until the internal temperature reached 70 °C. After cooking, the chop was cooled to approximately 25 °C and then weighed. Cooking loss was presented as a percentage of the initial weight. Three cores (1.3 cm in diameter) removed parallel to the orientation of the muscle fiber were obtained from each cooked chop for measurement of Warner–Bratzler shear force (WBSF). Shear force was measured by cutting the core vertically to the muscle fiber orientation using an

ACCEPTED MANUSCRIPT

Instron universal testing system (Model 4400, Instron Corp., Norwood, MA, USA) with a blade speed of 3.00 mm/s and a load cell capacity of 50 kg. WBSF (N) was presented as the average of the shear force value for the three cores.

T

2.6. Statistical analysis

IP

A total of 1147 peptides and 138 proteins were identified. Among these proteins, 39

CR

proteins that had a score > 39 were selected to compare their LFQ intensity among the LTL

US

regions. Protein scores (−10 × log(P)) were based on the calculated probability (P) that the observed match between the experimental data and the data-based sequence was a random event.

AN

All experimental data including LFQ, immunohistochemistry, and meat quality characteristics were presented as means ± SE. The statistical analysis was performed using a SAS program (9.4,

M

SAS Institute, USA). Pearson correlation coefficients were determined for relationships between

ED

proteome, meat quality, and muscle fiber characteristics within each region of the porcine LTL

PT

muscle. The significance of the correlation was accepted at P < 0.05. For further analysis of relationships between proteome constituents and meat quality, a stepwise multiple linear

CE

regression was performed within each region of the porcine LTL muscle. The significance level

AC

for entry or removal of independent variables in the models was P < 0.05 (SAS 9.4).

3. Results 3.1. Intramuscular meat quality variation in porcine LTL muscle

ACCEPTED MANUSCRIPT

As shown in Table 1, instrumental color (CIE L* , a* , and b* ), drip loss, and WBSF showed significant (P < 0.05) differences among the regions; however, the other parameters were not significantly (P > 0.05) different. For surface color of pork chop, CIE L* and b* were higher in the anterior than in the medial and posterior regions, whereas CIE a* was higher in the

IP

T

medial region (P < 0.05). As shown in Fig. 1, chop surface color appeared reddish-pink in the

CR

anterior and medial regions but changed to pale and grayish-pink toward the posterior region. The medial region of LTL muscle was redder than the anterior and posterior regions. Drip loss

US

was significantly (P < 0.05) higher in the medial than in the anterior and posterior regions, whereas drip loss in the anterior and posterior regions was not significantly (P = 0.072) different.

AN

The medial region showed the highest WBSF, and the anterior region had the lowest WBSF

M

among the LTL groups (P < 0.05).

ED

3.2. Intramuscular proteome variation in porcine LTL muscle

PT

A total of 39 proteins based on the unique peptides for individual proteins were identified in porcine LTL muscle, and they are presented in Table 2. Several proteins, such as elongation

CE

factor 1-alpha (EEF1A1), calsequestrin (CASQ1), troponin C (fast skeletal muscle; TnC-fast),

AC

alpha-crystallin B chain (CRYAB), muscle 6-phosphofructokinase (PFKM), and glycerol-3phosphate dehydrogenase (GPD1), had low sequence coverage (below 30.0%). However, most proteins were identified with high sequence coverage, and the maximum was 91.5% (triosephosphate isomerase; TPI1). Twenty proteins are responsible for energy metabolism regardless of metabolic pathway (Fig. 2A). Specifically, 13 proteins are enzyme proteins responsible for glycolysis or gluconeogenesis, whereas 17 proteins are responsible for NADH or NAD metabolic processes (Fig. 2A and 2B). The others are mostly myofibrillar proteins

ACCEPTED MANUSCRIPT

including the myosin complex and myosin heavy chain and light chain isoforms. Among the myofibrillar proteins, myosin heavy chain isoforms 1 (myosin-1), 2 (myosin-2), and 4 (myosin4), myosin light chains 2 (MLC2) and 1 (fast; MLC1f), TnC-fast, tropomyosin alpha-1 (TPM1), and beta-tropomyosin (TPM2) were detected as abundant proteins in the anterior region (P <

IP

T

0.05; Fig. 3). However, other myofibrillar proteins such as myosin-7, myosin light chain 3

CR

(MLC3), and TPM2 were largely expressed in both medial and posterior regions (P < 0.05). Proteins responsible for glycolysis or gluconeogenesis such as phosphoglucomutase 1 (PGM1),

US

fructose-bisphosphate aldolase A (ALDOA), glucose-6-phosphate isomerase (GPI), phosphoglycerate mutase (PGAM2), phosphoglycerate kinase 1 (PGK1), and pyruvate kinase

AN

(PK-M), were highly expressed in both medial and posterior regions (P < 0.05). Chaperone

M

proteins identified in this study were CASQ1 and heat shock 27 kDa (Hsp27), which were highly

ED

expressed in the anterior region (P < 0.05). In addition, carbonic anhydrase 3 (CA3), which is involved in cellular stress responses, serum albumin (ALB), and hemoglobin subunits (alpha and

PT

beta; Hbα and Hbβ), which are responsible for oxygen transport, were also highly abundant in the anterior region (P < 0.05). However, myoglobin (Mb), an intramuscular oxygen transporter,

CE

was more highly expressed in the medial than in the anterior and posterior regions (P < 0.05).

AC

3.3. Muscle fiber characteristics As shown in Fig. 4A, four types of muscle fiber could be distinguished through immunohistochemistry analysis with monoclonal anti-myosin. Regardless of fiber type, muscle fiber size (CSA) was lowest in the anterior region (P < 0.05; Fig. 4B). CSA of type IIX and IIB was higher in the posterior than in the medial region (P < 0.05), whereas CSA of type I and IIA was not significantly different between the two groups (P > 0.05). Relative area (%) of type I

ACCEPTED MANUSCRIPT

was higher in the posterior region (Fig. 4C); however, type IIA and IIB presented higher relative area in the anterior region compared with that in the posterior region (P < 0.05). Muscle fiber type IIB was not significantly different in relative area among the LTL regions (P > 0.05). Fiber density of type IIA, IIX, and IIB was the highest in the anterior among the LTL regions (P < 0.05;

IP

T

Fig. 4D). The posterior region showed the lowest (P < 0.05) fiber density of type IIX and IIB;

CR

however, type IIA fiber density was lowest (P < 0.05) in the medial region. On the other hand, muscle fiber type I did not show any significant difference (P > 0.05) in fiber density among the

US

LTL groups.

AN

3.4. Relationship between proteome, meat quality, and muscle fiber characteristics Correlation coefficients between proteome components and meat quality traits involving

M

different regions of porcine LTL muscle are presented in Table 3. In the anterior region,

ED

contractile proteins such as myosin-1, MLC2, TPM2, MLC1f, and myosin-2 were positively

PT

correlated with CIE L* (P < 0.05), whereas enzymes responsible for glycolysis such as PGM1, LDHA, ALDOA, enolase 3, and TPI1 were negatively correlated with CIE L* (P < 0.05).

CE

Conversely, those proteins, except for ALDOA and TPI1, showed opposite trends with pH and WBSF. CIE a* was positively correlated with PGM1, but negatively correlated with MLC2 and

AC

myosin-2 (P < 0.05). CIE b* was positively correlated with myozenin-1 and PYGM but negatively correlated with LDHA and GPD1 (P < 0.05). In the medial region, LDHA, PGM1, and MLC3 were negatively correlated with drip loss, whereas CA3, myosin-2, myosin-1, Hsp27, and TPM2 were positively correlated with drip loss (P < 0.05). PGM1, CA3, and myosin-1 in the medial region exhibited the same trend with CIE L* , pH, and WBSF in the anterior region. CIE a* was correlated with GPD1 (r = -0.52; P < 0.01). In the posterior region, TPM2, myosin-1,

ACCEPTED MANUSCRIPT

myosin-2, and Hsp27 were negatively correlated with cooking loss, whereas LDHA showed positive correlation (P < 0.01) with cooking loss. CIE a* and WBSF were negatively correlated with CA3 and myosin-1 (P < 0.05), whereas drip loss was positively correlated with GPD1 (P < 0.01). Regardless of region, CIE L* was positively correlated with myosin-1, Hsp27, and CA3,

CR

negative correlation with pH throughout the porcine LTL muscle.

IP

T

while WBSF was negatively correlated with myosin-1 and CA3. In addition, myosin-1 showed

The correlation coefficients between proteome and muscle fiber characteristics within regions of

US

porcine LTL muscle are shown in Table 4. The correlation trends of proteome with relative area

AN

or CSA were in the opposite direction (positive vs. negative) of the r-value between slow-twitch fiber type (I) and fast-twitch fiber types (IIA, IIX, and IIB). However, all fiber types in CSA had

M

similar correlation trends with proteome regardless of the porcine LTL region. Among the

ED

proteins, Hsp 27 and contractile proteins such as myosin-1, TPM2, MLC1f, and myosin-2 were negatively correlated with relative area of type I and CSA of type I, IIX, and IIB (P < 0.05).

PT

However, these proteins were positively correlated with the relative area of type IIA and IIX, and

CE

fiber density of all fast-twitch types (P < 0.05). Metabolic enzymes such as LDHA, PGM1, and enolase 3 showed positive correlations with relative area of type I but negative correlations with

AC

relative area of type IIA or IIX and with fiber density of fast-twitch fiber types (P < 0.05). This trend was also found in the medial region. Relative area of type IIB did not have any correlations with any proteins in the anterior as well as medial regions; however, several correlations were found involving the posterior region. In other words, TPM2, CA3, MLC1f, MLC2, myosin-1, and Hsp27 were negatively correlated with relative area of type IIB, whereas troponin I (fasttwitch skeletal; TnI-fast) was positively correlated with relative area of type IIB (P < 0.05).

ACCEPTED MANUSCRIPT

Common trends among three regions of porcine LTL muscle were found for myosin-1, TPM2, Hsp27, myosin-2, MLC2, PGAM2, and LDHA. To understand the relationship between intramuscular variation in meat quality and proteome,

T

regression models within regions of muscle were established using meat quality traits as

IP

dependent, and proteins as independent variables (Table 5). Increased CIE L* was predicted by

CR

the elevation of myosin-1, CA3, and Hsp27 regardless of muscle regions (P < 0.05). Myosin-1 and CA3 also exhibited robust models for WBSF but both had negative coefficients (P < 0.05).

US

The pH value was predicted to decrease with increase in myosin-1 (P < 0.05), regardless of

AN

muscle region. Unlike CIE L*, WBSF, and pH, the models commonly predicted in three different regions were not obtained in CIE a* and b* , drip loss, and cooking loss. However, CIE

M

a* was negatively correlated with myosin-1 in the anterior as well as posterior regions. Increased

ED

cooking loss was predicted with elevated LDHA in the posterior region (P < 0.05). Significant coefficients were predicted for drip loss with LDHA, PGM1, myosin-1, myosin-2, Hsp27, and

PT

TPM2 in the medial region, whereas only GAPDH exhibited a significant model involving drip

CE

loss in the posterior region (P < 0.01). Although the anterior region did not have any predicted models involving drip and cooking losses, other meat quality traits were explained with various

AC

proteins. In particular, myosin-1, myosin-2, MLC2, TPM2, LDHA, PGM1, CA3, and Hsp27 were correlated with CIE L* , pH or WBSF in the anterior region. However, models for meat quality traits in the medial and posterior regions were predicted with relatively few proteins.

4. Discussion

ACCEPTED MANUSCRIPT

Remarkable intramuscular variation of meat quality was found in porcine LTL muscle. Specifically, meat color, water-holding capacity, and tenderness differed between the anterior, medial, and posterior regions. As shown in Fig. 1, meat color changed from reddish-pink in the anterior region to pale and grayish-pink in the posterior region. The lower CIE L* , a* , and b*

IP

T

values in the posterior region than in the anterior or medial regions were supported by the visual

CR

assessment. The highest redness and Mb content were found in the medial region despite of no correlation between redness and Mb content being identified in this study. It is known that high

US

abundance of Mb is closely related to red color with high CIE a * value (Bekhit & Faustman, 2005; Kim, Jeong, Hur, Yang, Jeon, & Joo, 2010). In previous studies, intramuscular color

AN

variations have mostly been found in SM and ST muscles. For example, bovine SM is classified

M

in two parts according to the color stability: color-stable (outside) and color-labile (inside). Nair

ED

et al. (2016) have demonstrated that greater abundance of glycolytic enzymes leads to rapid postmortem glycolysis in outside SM and consequently causes lower stability of meat color.

PT

Porcine SM also comprises two distinct parts (dark and light), and the dark part had lower lightness and higher Mb content; however, redness was not different between the two parts (Kim

CE

et al., 2018). Porcine ST muscle exhibits intramuscular variation in color caused by the

AC

differential expression of myosin isoforms and muscle fiber type distribution (Kim et al., 2018; Lefaucheur et al., 1995). In porcine LTL muscle, the color of the anterior region could be distinguished from those of the other regions with highly expressed proteins such as myosin-1, myosin-2, TPM2, and Hsp27, which were positively related to CIE L* or b* . On the other hand, enolase 3, ALDOA, LDHA, PGM1, and TPI1 which were negatively related to CIE L* , could be markers for different color between the anterior and medial or between the anterior and posterior regions. Despite a similar abundance of proteome between the medial and posterior regions,

ACCEPTED MANUSCRIPT

these two regions could be distinguished by GAPDH, which is abundant in the medial region and negatively related to CIE a* in the medial region. In a previous study, association of Hsp27, alpha crystalline, hemoglobin, succinate dehydrogenase, NADH dehydrogenase, and ATPase beta subunit with lightness has been demonstrated in porcine SM muscle (Sayd et al., 2006).

IP

T

High composition of fast twitch-glycolytic fiber types (IIX and IIB) is closely related to high

CR

abundance of enzyme proteins responsible for glycolysis, and this relationship could explain intramuscular color variation of porcine SM and ST muscles. However, in the present study, not

US

only relative area composition but also muscle fiber size or fiber density were closely related to

AN

differentially expressed proteome in porcine LTL muscle.

Intramuscular variation of meat color as well as tenderness is also related to CSA of

M

muscle fiber regardless of type. Except for metabolic enzymes, chaperone (Hsp27) and

ED

myofibrillar proteins (myosin-1, MLC1f, MLC2, myosin-2, and TPM2) were expressed at higher levels in the anterior region than in other regions. These proteins were negatively related to the

PT

WBSF in the anterior region, which is the most tender, whereas the WBSF in the medial and

CE

posterior regions was predicted with only myosin-1 among those proteins. In general, muscles highly composed of large-size muscle fibers tend to be tough; fast-twitch glycolytic fiber types

AC

especially increase the toughness of meat (Karlsson, Enfalt, Essen-Gustavsson, Lundstrom, Rydhmer, & Stern, 1993; Maltin, Warkup, Mattews, Grant, Porter, & Delday, 1997). Our results agreed with previous reports demonstrating that the anterior region has the smallest fibers regardless of fiber type but has the highest relative area of type IIX. However, the proteins composing the myosin complex, especially the fast-twitch type of myosin isoforms (myosin-1, myosin-2, and myosin-4), are abundant in the anterior region. Among the fast-twitch fibers including IIA, IIX, and IIB, type IIX and IIB predominantly rely on energy produced by

ACCEPTED MANUSCRIPT

glycolysis, and thus those fibers contain various enzymes responsible for glycolysis (Kim et al., 2018; Nader & Esser, 2001). In the present study, metabolic enzymes did not show the consistency with contractile proteins within porcine LTL muscle, when compared to myosin isoforms (1, 2, 4, and 7) and glycolytic enzymes between anterior, medial, and posterior regions,

IP

T

as shown in Fig. 3. In addition, a few proteins such as LDHA, PGM1, and GPD1, exhibited

CR

positive coefficients with drip loss in the medial region. These proteins influenced water-holding capacity because rapid postmortem glycolysis is associated with abundant glycolytic enzymes,

US

resulting in low ultimate pH and water-holding capacity (Bee et al., 2007; Ryu & Kim, 2006; te Pas et al., 2009). In the present study, intramuscular variations of water-holding capacity and

AN

tenderness were found in porcine LTL muscle; however, pH was not different among the LTL

M

regions. This tendency was consistent with our previous study, which demonstrated

ED

intramuscular variations of muscle fiber characteristics and proteome in porcine SM and ST muscles (Kim et al., 2018). The dark part of SM, which has higher Mb content and oxidative

PT

fiber types, presented higher drip loss than that of the light part, even though proteins associated with glycolysis are less expressed in the dark part than in the light part. Moreover, pH was not

CE

different between the two parts of SM, consistent with the result in LTL muscle. The pH is

AC

associated with myosin-1, however, other contractile proteins, such as myosin-2, MLC2, and TPM2 and stress response proteins (Hsp27 and CA3) as well as glycolytic enzymes, including PGM1, LDHA, and TPI1, allow distinction of the anterior region from the medial and posterior regions in terms of muscle pH. Two protein groups (contractile proteins and associated vs. glycolysis and gluconeogenesis) could be distinguished through protein–protein functional interactions (Fig. 2B). The enzymes associated with glycolysis or gluconeogenesis presented a similar relationship

ACCEPTED MANUSCRIPT

with meat quality traits; however, among the contractile proteins, proteins associated to fast-

T

twitch fibers were closely related to meat quality traits, especially, meat color and tenderness.

IP

5. Conclusions

CR

Intramuscular variations of meat quality including meat color, drip loss, and WBSF were

US

found in porcine LTL muscle. These variations resulted from the differential abundance of proteome and different muscle fiber size and density regardless of type. The reddish-pink color

AN

of the anterior region changed to pale and grayish-pink in the posterior region, which could be

M

explained by differential abundance of enolase 3, ALDOA, LDHA, PGM1, and TPI1 in the medial and posterior regions and by highly expressed GAPDH in the medial region. Low water-

ED

holding capacity in the medial region was associated with the greater abundance of glycolysis

PT

enzymes, whereas relatively higher tenderness in the anterior region could be explained by the greater abundance of Hsp27, MLC1f, MLC2, myosin-2, myosin-1, TPM2 as well as TPI1.

CE

Therefore, the longitudinal variations of color, water-holding capacity, and tenderness in porcine

AC

LTL muscle are associated with differential abundance of proteins, especially contractile proteins, glycolytic enzymes, and Hsp27.

Acknowledgments This study was supported by National Research Foundation (NRF2016R1D1A1B03935656) funded by the Ministry of Education, Republic of Korea.

ACCEPTED MANUSCRIPT

References AOAC (1995). Official method of analysis (16th ed.) Washington, DC: Association of the Official Analytical Chemists.

T

Bekhit, A. E. D., & Faustman, C. (2005). Metmyoglobin reducing activity. Meat Science, 71,

IP

407–439.

CR

Bee, G., Anderson, A. L., Lonergan, S. M., & Huff-Lonergan, E. (2007). Rate and extent of pH decline affect proteolysis of cytoskeletal proteins and water-holding capacity in pork. Meat

US

Science, 76, 359–365.

AN

Chang, K. C., Costa, N., Blackley, R., Southwood, O., Evans, G., Plastow, G., Wood, J. D., & Richardson, R. I. (2003). Relationships of myosin heavy chain fibre types to meat quality

M

traits in traditional and modern pigs. Meat Science, 64, 93–103.

ED

CIE (Commission Internationale de l’Eclairage). (1976). Recommendations on uniform color spaces-color difference equations, Psychometric Color Terms. Supplement No. 2 to CIE

PT

Publication No. 15 (E-1.3.1.) 1978, 1971/(TC-1-3). Commission Internationale de

CE

l’Eclairage, Paris, France.

Cox, J., & Mann, M. (2008). MaxQuant enables high peptide identification rates, individualized

AC

p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnology, 26, 1367–1372 Eggert, J. M., Depreux, F. F. S., Schinckel, A. P., Grant, A. L., & Gerrard, D. E. (2002). Myosin heavy chain isoforms account for variation in pork quality. Meat Science, 61, 117–126. Folch, J., Lees, M., & Sloane-Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497– 509.

ACCEPTED MANUSCRIPT

Honikel, K. O. (1987). How to measure the water-holding capacity of meat? Recommendation of standardized methods. In: P. V. Tarrant, G. Eikelenboom, and G. Monin, editors, Evaluation and control of meat quality in pigs. Martinus Nijhoff, Dordrecht, The Netherlands. p. 129– 142.

IP

T

Karlsson, A., Enfalt, A. C., Essen-Gustavsson, B., Lundstrom, K., Rydhmer, L., & Stern, S.

CR

(1993). Muscle histochemical and biochemical properties in relation to meat quality during selection for increased lean tissue growth rate in pigs. Journal of Animal Science, 71, 930–

US

938.

Karlsson, A. H., Klont, R. E., & Fernandez, X. (1999). Skeletal muscle fibres as factors for pork

AN

quality. Livestock Production Science, 60, 255–269.

M

Kim, G. D., Jeong, J. Y., Hur, S. J., Yang, H. S., Jeon, J. T., & Joo, S. T. (2010). The relationship

ED

between meat color (CIE L* and a* ), myoglobin content, and their influence on muscle fiber characteristics and pork quality. Korean Journal for Food Science of Animal Resources, 30,

PT

626–633.

Kim, G. D., Ryu, Y. C., Jeong, J. Y., Yang, H. S., & Joo, S. T. (2013). Relationship between

CE

pork quality and characteristics of muscle fibers classified by the distribution of myosin

AC

heavy chain isoforms. Journal of Animal Science, 91, 5525–5534. Kim, G. D., Yang, H. S., & Jeong, J. Y. (2018). Intramuscular variations of proteome and muscle fiber type distribution in semimembranosus and semitendinosus muscles associated with pork quality. Food Chemistry, 244, 143–152. Konhilas, J. P., Irving, T. C., & de Tombe, P. P. (2002). Length-dependent activation in three striated muscle types of the rat. Journal of Physiology, 544, 225–236.

ACCEPTED MANUSCRIPT

Kwasiborski, A., Sayd, T., Chambon, C., Santé-Lhoutellier, V., Rocha, D., & Terlouw, C. (2008). Pig longissimus lumborum proteome: Part II: Relationships between protein content and meat quality. Meat Science, 80, 982–996.

IP

type formation in the pig. Developmental Dynamics, 203, 27–41.

T

Lefaucheur, L., Edom, F., Ecolan, P., & Butler-Browne, G. S. (1995). Pattern of muscle fiber

CR

Maclntosh, B. R., Gardiner, P. F., & McComas, A. (2005). Muscle architecture and muscle fiber anatomy. In Skeletal muscle form and function (pp. 3‒ 21). USA: Human Kinetics.

US

Maltin, C. A., Warkup, C. C., Mattews, K. R. C., Grant, M. A., Porter, D., & Delday, M. I.

AN

(1997). Pig muscle fibre characteristics as a source of variation in eating quality. Meat Science, 47, 237–248.

M

McDonald, K. S., Wolff, M. R., & Moss, R. L. (1997). Sarcomere length dependence of the rate

ED

of tension redevelopment and submaximal tension in rat and rabbit skinned skeletal muscle fibres. Journal of Physiology, 501, 607–621.

PT

Nader, G. A., & Esser, K. A. (2001). Intracellular signaling specificity in skeletal muscle in

CE

response to different modes of exercise. Journal of Applied Physiology, 90, 1936–1942. Nair, M. N., Suman, S. P., Chatli, M. K., Li, S., Joseph, P., Beach, C. M., & Rentfrow, G. (2016).

AC

Proteome basis for intramuscular variation in color stability of beef semimembranosus. Meat Science, 113, 9–16. Park, B. Y., Kim, N. K., Lee, C. S., & Hwang, I. H. (2007). Effect of fiber type on postmortem proteolysis in longissimus muscle of Landrace and Korean native black pigs. Meat Science, 77, 482–491.

ACCEPTED MANUSCRIPT

Promeyrat, A., Sayd, T., Laville, E., Chambon, C., Lebret, B., & Gatellier, P. (2011). Early postmortem sarcoplasmic proteome of porcine muscle related to protein oxidation. Food Chemistry, 127, 1097–1104. Ryu, Y. C., & Kim, B. C. (2006). Comparison of histochemical characteristics in various pork

IP

T

groups categorized by postmortem metabolic rate and pork quality. Journal of Animal

CR

Science, 84, 894–901.

Sayd, T., Morzel, M., Chambon, C., Franck, M., Figwer, P., Larzul, C., et al. (2006). Proteome

US

analysis of the sarcoplasmic fraction of pig semimembranosus muscle: Implications on meat color development. Journal of Agricultural & Food Chemistry, 54, 2732–2737.

AN

te Pas, M. F. W., Jansen, J., Broekman, K. C. J. A., Reimert, H., & Heuven, H. C. M. (2009).

M

Postmortem proteome degradation profiles of longissimus muscle in Yorkshire and Duroc

ED

pigs and their relationship with pork quality traits. Meat Science, 8, 744–751. te Pas, M. F. W., Kruijt, L., Pierzchala, M., Crump, L. E., Boeren, S., Keuning, E., et al. (2013).

PT

Identification of proteomic biomarkers in M. Longissimus dorsi as potential predictors of

CE

pork quality. Meat Science, 95, 679–687. van de Wiel, D. F. M., & Zhang, W. L. (2007). Identification of pork quality parameters by

AC

proteomics. Meat Science, 77, 46–54.

ACCEPTED MANUSCRIPT

Figure Legends Fig. 1. Representative photos (whole and chops) of porcine longissimus thoracis et lumborum muscle.

1-15

Numbers on chops indicate serial numbers designated anterior (1) to

T

posterior (15).

IP

Fig. 2. Protein–protein interactions and representative biological functions of differentially

CR

expressed proteins between anterior, medial, and posterior porcine longissimus thoracis et

US

lumborum muscle. (A) Major biological processes and molecular functions are represented. Protein ID is presented by gene (same as in Table 2). (B) Network shows interactions, and edges

AN

represent protein–protein associations (see legend).

M

Fig. 3. Differentially expressed proteins between anterior, medial, and posterior regions of

ED

porcine longissimus thoracis et lumborum (LTL) muscles. Different letters (a–c) on the bar indicate significant (P < 0.05) differences between anterior, medial, and posterior regions of

PT

porcine LTL muscle.

CE

Fig. 4. Results of immunohistoche mistry of porcine longissimus thoracis et lumborum muscle. (A) Transverse serial sections stained with four monoclonal antibodies. *, type I; •, type

AC

IIA; o, type IIX; Δ, type IIB. Bar = 100 µm. (B) Relative area composition (%); (C) crosssectional area (µm2 ); (D) fiber density (number/mm2 ). Different letters (a–c) on the bar indicate significant (P < 0.05) differences between anterior, medial, and posterior regions within the same fiber types.

ACCEPTED MANUSCRIPT

Table 1. Comparison of meat quality characteristics between the anterior, medial, and posterior regions of porcine longissimus thoracis et lumborum muscles Medial

Posterior

pH Crude fat (%)

5.54±0.11 1.63±0.28

5.53 ±0.08 1.40 ±0.14

5.58±0.13 1.52±0.15

Moisture (%)

74.10±0.08

74.20 ±0.28

74.10±0.12

Crude protein (%) CIE L*

23.81±0.42 53.68±2.33a

23.90 ±0.14 51.62 ±2.18b

23.74±0.14 51.74±1.08b

CIE a*

4.03±0.98b

4.69 ±0.91a

3.83±0.49b

CIE b* Drip loss (%)

7.27±1.24a 2.42±0.28b

6.47 ±0.99b 3.80 ±0.35a

6.51±0.62b 2.67±0.16b

29.07 ±3.46

31.57±2.40

Cooking loss (%)

33.81±2.55

a

49.49 ±2.55

US

c

42.43±5.78b

CE

PT

ED

M

AN

Means±SE with same superscripts are not significantly (P > 0.05) different in the same row.

AC

a-c

IP

28.06±0.28

Warner-Bratzler shear force (N)

T

Anterior

CR

Meat quality traits

ACCEPTED MANUSCRIPT

Table 2. Proteins identified in porcine longissimus thoracis et lumborum muscle by LC-MS/MS Number of unique Sequence coverage (%) peptides Protein name Gene Score2) Anterio Posterio Anterio Posterio Medial Medial r r r r P68137 Actin, alpha skeletal muscle ACTA 33 35 34 87.3 88.6 88.6 323.3 1 A1X899 Beta-tropomyosin TPM2 2 2 2 41.5 41.5 43.0 226.1 A1XQT6 MLC1f MYL1 4 4 4 93.2 93.2 93.2 323.3 Q5XLD2 MLC2 MYLP 21 17 16 95.3 88.8 82.8 323.3 F F1SNW4 MLC3 MYL3 3 3 3 30.5 36.0 36.0 67.0 Q9TV61 Myosin-1 MYH1 14 13 12 68.4 66.8 62.6 323.3 Q9TV63 Myosin-2 MYH2 8 6 5 53.0 51.7 46.9 89.3 Q9TV62 Myosin-4 MYH4 32 37 36 69.2 68.6 67.9 323.3 P79293 Myosin-7 MYH7 32 31 32 32.4 30.9 31.8 323.3 Q4PS85 Myozenin-1 MYO 6 5 5 53.7 47.0 47.0 151.8 Z1 P42639 Tropomyosin alpha-1 chain TPM1 10 9 9 55.6 55.6 54.9 323.3 A1XQV5 Troponin C, fast skeletal TNNC 4 4 3 22.6 22.6 22.6 179.3 muscle 2 Q4JH15 Troponin I, fast-twitch skeletal TNNI 9 9 5 53.3 53.3 44.5 67.2 2 Q75NH0 Troponin T, fast skeletal TNNT 3 3 2 45.6 45.6 41.2 242.6 muscle 3 Q1KYT0 Enolase 3 (beta) ENO3 21 22 21 65.9 71.9 72.6 323.3 Q6UV40 Fructose-bisphosphate aldolase ALDO 4 4 4 56.9 56.9 56.9 191.9 A A P08059 Glucose-6-phosphate GPI 16 16 17 40.7 43.7 40.9 323.3 isomerase P00355 Glyceraldehyde-3-phosphate GAPD 23 23 22 82.0 79.6 79.6 323.3 dehydrogenase H D2XN65 Glycerol-3-phosphate GPD1 2 2 2 29.8 21.5 26.9 74.5 dehydrogenase P00339 L-lactate dehydrogenase A LDHA 22 23 23 80.4 81.6 85.5 323.3 chain Q1HL06 Muscle 6-phosphofructokinase PFKM 4 4 4 26.6 26.6 25.4 295.1 Q7SIB7 Phosphoglycerate kinase 1 PGK1 1 1 1 59.7 78.7 71.0 323.3 B5KJG2 Phosphoglycerate mutase PGA 16 17 15 83.8 83.8 83.4 301.5 M2 F1SHL9 Pyruvate kinase PKM 27 29 26 67.2 67.2 66.9 323.3 Q29371 Triosephosphate isomerase TPI1 21 24 22 91.5 91.5 91.1 323.3 Q19PY1 Alpha-1,4 glucan PYG 2 2 2 66.9 70.9 66.9 120.5 phosphorylase M Q9TQR6 Phosphoglucomutase 1 PGM1 30 31 31 62.1 63.7 63.3 323.3 Q8WMV Ca2+ ATPase of fast twitch 1 ATP2 24 20 15 31.2 24.4 23.5 312.0 7 skeletal muscle sarcoplasmic A1 Accessio n no.1)

1)

AC

CE

PT

ED

M

AN

US

CR

IP

T

1)

ACCEPTED MANUSCRIPT

reticulum F1RJW7 Calsequestrin

AC

CE

PT

ED

M

AN

US

CR

IP

T

CASQ 3 3 3 15.7 12.2 12.2 33.5 1 Q7M2W Alpha-crystallin B chain CRYA 4 4 3 25.7 25.7 18.3 38.7 6 B Q5S1U1 Heat shock 27 kDa protein HSPB 6 5 4 55.6 47.3 39.6 62.1 1 Q5XLD3 Creatine kinase M-type CKM 37 38 39 74.3 75.6 75.9 323.3 Q5S1S4 Carbonic anhydrase 3 CA3 20 19 18 79.6 79.6 79.6 323.3 P00571 Adenylate kinase isoenzyme 1 AK1 13 13 13 66.5 66.5 66.5 151.7 B9W5V0 Elongation factor 1-alpha EEF1 2 3 3 7.6 14.7 14.7 41.8 A1 P01965 Hemoglobin subunit alpha HBA1 4 4 3 44.0 44.0 55.3 31.3 P02067 Hemoglobin subunit beta HBB 6 6 6 67.3 67.3 67.3 138.6 F1RUN2 Serum albumin ALB 15 14 14 35.6 33.6 33.4 323.3 P02189 Myoglobin MB 14 12 12 85.1 74.7 85.1 237.4 1) Accession no., protein name, and gene were derived from UniProt database, taxonomy Sus scrofa 9823 (48,896 sequences). 2) Scores > 39 indicate identity (P < 0.05).

ACCEPTED MANUSCRIPT

Table 3. Correlation coefficients between proteome and meat quality traits in different regions of porcine longissimus thoracis et lumborum muscle

0.42*

-0.41

*

0.41 0.43*

0.52** 0.38* -0.43* -0.43* -0.51** -0.44*

-0.35*

-0.39*

-0.41*

-0.39*

0.35*

0.35* 0.39* -0.42* 0.39*

US

0.43*

0.44* 0.43* -0.36* -0.35* -0.46* -0.43*

IP

-0.35* -0.40*

*

M

AN

-0.45

-0.36* -0.40* 0.45* -0.40* 0.36*

-0.43*

-0.36*

*

-0.35

0.38

-0.36 -0.52**

*

*

0.36

*

-0.35 0.37* 0.39* 0.38*

0.47* 0.36* 0.38* 0.40*

0.56** 0.52** -0.37*

*

*

PT

CE

AC

*

WarnerDrip Bratzler shear loss force

CR

-0.54** -0.42* 0.44* 0.44* 0.54** 0.47* -0.35* 0.41*

ED

Anterior Phosphoglucomutase 1 L-lactate dehydrogenase A chain Carbonic anhydrase 3 MLC2 Myosin-1 Beta-tropomyosin Fructose-bisphosphate aldolase A MLC1f Myozenin-1 Alpha-1,4 glucan phosphorylase Myosin-2 Glycerol-3-phosphate dehydrogenase Enolase 3 (beta) Troponin I, fast-twitch skeletal Heat shock 27 kDa protein Calsequestrin Triosephosphate isomerase Medial L-lactate dehydrogenase A chain Phosphoglucomutase 1 MLC3 Glycerol-3-phosphate dehydrogenase Carbonic anhydrase 3 Myosin-2 Myosin-1 Heat shock 27 kDa protein Beta-tropomyosin Posterior Beta-tropomyosin Carbonic anhydrase 3 Glycerol-3-phosphate dehydrogenase Heat shock 27 kDa protein L-lactate dehydrogenase A chain MLC2 MLC3 Myosin-1 Myosin-2 * , P < 0.05; ** , P < 0.01.

Cooking loss

pH

T

CIE L* CIE a* CIE b*

Proteins

0.43 0.51** 0.56** 0.54** 0.54**

-0.46*

0.43* -0.41* -0.36* -0.43*

-0.47* -0.36*

-0.37* **

0.57 0.42* -0.36* 0.35* -0.36* 0.50*

-0.48* 0.53** -0.38*

-0.36*

-0.45* -0.46*

-0.46*

ACCEPTED MANUSCRIPT

Table 4. Correlation coefficients between proteome and muscle fiber characteristics in different regions of porcine longissimus thoracis et lumborum muscle Proteins

Relative area IIA IIX IIB

I

Cross-sectional area I IIA IIX IIB

I

Fiber density IIA IIX IIB

Anterior

-0.36* 0.41* 0.38* 0.41* 0.36* * -0.50 0.47* 0.50*

-0.38* -0.36* * -0.45 0.51** * 0.35 0.38* -0.45* -0.50*

-0.37* 0.37* 0.37* -0.48* 0.40*

AC

Phosphoglucomutase 1

Fructose-bisphosphate aldolase A Enolase 3 (beta) Glucose-6-phosphate isomerase MLC3 Myosin-7 Glycerol-3-phosphate dehydrogenase Elongation factor 1-alpha Carbonic anhydrase 3 MLC2

*

IP

CR

-0.38

-0.48* 0.35* 0.45* 0.37* 0.42* -0.39

*

-0.36

-0.38* -0.43 -0.43*

0.41* 0.40* 0.41* 0.45* 0.45*

-0.37*

-0.44* -0.48*

0.43* 0.50* 0.50*

0.38* 0.41*

-0.37* -0.43* -0.43* -0.39*

*

*

*

-0.39

0.53** -0.35*

0.52** 0.38* -0.35* * -0.48 -0.37*

0.43* 0.38* 0.50* 0.52**

0.40*

0.36*

PT

-0.36* -0.36* 0.46 0.53** 0.53**

*

- 0.38 0.48 0.51** -0.39*

-0.40*

-0.35* -0.40* -0.40* 0.48* 0.52** 0.53** -0.35*

*

-0.39* *

CE

Calsequestrin Medial L-lactate dehydrogenase A chain Phosphoglycerate mutase

-0.38*

0.39* -0.36* * * -0.50 0.40 0.48*

*

Heat shock 27 kDa protein

T

0.41* 0.35* 0.49* 0.52**

- -0.50* -0.50* 0.54** -0.43* 0.55** 0.55** 0.44* 0.40* 0.41* 0.44* 0.38* 0.38* 0.54** 0.54** 0.54**

US

Phosphoglycerate mutase Beta-tropomyosin Fructose-bisphosphate aldolase A MLC1f Alpha-1,4 glucan phosphorylase Myosin-2 Glycerol-3-phosphate dehydrogenase Enolase 3 (beta) Troponin I, fast-twitch skeletal MLC3

0.54** -0.33* -0.49*

AN

Myosin-1

0.41* 0.47*

M

L-lactate dehydrogenase A chain Carbonic anhydrase 3 MLC2

0.44* -0.50* -0.46*

ED

Phosphoglucomutase 1

*

-0.46 -0.50

-0.38* -0.44*

0.42* 0.39*

0.55** 0.54** -0.38* -0.43* -0.42* - -0.44* -0.41* 0.55** -0.36* -0.44* -0.41*

0.36* 0.43 0.46 0.42 0.43*

-0.37* -0.37* -0.39 -0.40* -0.37*

0.40* 0.40* 0.42* 0.44* 0.38* 0.36* 0.42*

*

*

*

-0.35*

*

0.40* 0.36*

0.36* -0.37*

-0.35*

*

0.38

*

-0.37

*

-0.35

0.40* 0.42* 0.36* 0.35*

ACCEPTED MANUSCRIPT -0.35* -0.39* -0.39* -0.40* -0.39* -0.43* -0.42*

*

-0.44

0.36

-0.44* -0.44*

0.46* 0.46*

0.38* 0.40* 0.38* 0.41* 0.42*

-0.39* -0.39* -0.35* 0.36* 0.44* 0.44* -0.41* -0.42* 0.37* 0.45* 0.44*

*

-0.37

-0.44* 0.50* 0.52** -0.49* 0.38* 0.51** -0.49* 0.38* 0.50*

-0.36* -0.40* -0.43* 0.50* 0.47* 0.47* -0.35 -0.44* -0.47* -0.38* 0.44* 0.51** 0.51** * * -0.42 -0.39 -0.49* -0.50* 0.47* 0.53** 0.51**

-0.44*

-0.44* -0.40* -0.45* -0.39* -0.36*

T

*

0.39*

0.42* 0.39*

-0.39* *

-0.38 -0.42*

0.35*

-0.42* 0.35*

*

0.35 0.42*

AN

0.35 0.41*

US

0.39* *

0.41* *

*

0.35 0.37 0.36* 0.43* 0.37* 0.44*

-0.38* -0.36* -0.35 -0.40* -0.44* *

-0.35* -0.36*

0.48* 0.51** 0.52** ** 0.52 0.53** -0.37* 0.53** 0.47* 0.50* 0.54** 0.40* -0.47* 0.52** 0.53** 0.55** 0.43* -0.38* -0.36* -0.39* -0.36* 0.39* 0.37* * * * * * 0.36 0.41 -0.45 -0.36 0.35 0.35* 0.35* * * * * * * * -0.44 0.49 0.46 -0.42 -0.45 -0.42 -0.41 0.56** 0.47* 0.51** ** 0.51 -0.43* 0.40* -0.44* -0.39* -0.40* -0.45* 0.41* 0.42* 0.46* 0.40* -0.35* -0.44* 0.39* 0.37* 0.42* 0.40* -0.36* -0.41* -0.41* * 0.35

ED

-0.49* 0.39* 0.50* -0.37* -0.50* -0.45* -0.47*

CE

L-lactate dehydrogenase A chain MLC1f MLC2

0.41*

PT

Heat shock 27 kDa protein

-0.44*

M

Ca2+ ATPase of fast twitch 1 skeletal muscle sarcoplasmic reticulum Carbonic anhydrase 3 Enolase 3 (beta) Fructose-bisphosphate aldolase A Glucose-6-phosphate isomerase Glycerol-3-phosphate dehydrogenase

- -0.39* 0.48* 0.50* 0.53** 0.53** * * * -0.44 -0.39 -0.40 -0.45* 0.41* 0.42* 0.46*

CR

-0.50* 0.41* 0.52** -0.38* -0.50* -0.42* -0.46*

Beta-tropomyosin

Myosin-1

*

IP

MLC1f Alpha-1,4 glucan phosphorylase Myosin-2 Ca2+ ATPase of fast twitch 1 skeletal muscle sarcoplasmic reticulum Myosin-1 Heat shock 27 kDa protein Beta-tropomyosin Posterior Alpha-1,4 glucan phosphorylase

AC

Myosin-2 Phosphoglycerate mutase Troponin I, fast-twitch skeletal * , P < 0.05; ** , P < 0.01.

Table 5. Result of multiple regression for relationship between meat quality traits and proteome in each portion of porcine longissimus thoracis et lumborum muscle

ACCEPTED MANUSCRIPT

Entered independent variable

Coefficient

SE

P-value

0.82**

Phosphoglucomutase 1 L-lactate dehydrogenase A chain Carbonic anhydrase 3 MLC2 Myosin-1 Beta-tropomyosin Fructose-bisphosphate aldolase A MLC1f Myosin-2 Enolase 3 (beta) Troponin I, fast-twitch skeletal Heat shock 27 kDa protein Calsequestrin Phosphoglucomutase 1 MLC2 Myosin-1 L-lactate dehydrogenase A chain Myozenin-1 Alpha-1,4 glucan phosphorylase Glycerol-3-phosphate dehydrogenase Phosphoglucomutase 1 L-lactate dehydrogenase A chain Carbonic anhydrase 3 MLC2 Myosin-1 Beta-tropomyosin Myosin-2 Enolase 3 (beta) Heat shock 27 kDa protein Triosephosphate isomerase Phosphoglucomutase 1 L-lactate dehydrogenase A chain Carbonic anhydrase 3 MLC2 Myosin-1 Beta-tropomyosin MLC1f Myosin-2 Troponin I, fast-twitch skeletal Heat shock 27 kDa protein Triosephosphate isomerase

-0.27

0.04

< 0.001

-0.82

0.27

0.011

0.47 0.89 3.18 0.07

0.14 0.26 0.50 0.02

0.006 0.006 < 0.001 0.003

0.11

0.045

0.87 0.35 -0.70 -0.30 0.17 -0.01 0.48 -1.61 -5.32

0.29 0.11 0.30 0.11 0.05 0.01 0.15 0.70 1.91

0.013 0.010 0.039 0.019 0.004 0.020 0.011 0.045 0.020

-4.01

1.38

0.016

0.18

0.06

0.015

0.33

0.10

0.009

-0.26

0.08

0.006

27.08

7.77

0.006

102.08

31.64

0.009

-46.28 -85.78 -333.58 -7.50 -40.73 82.47 -20.03 -19.87 1.10

19.42 37.81 87.16 2.26 14.01 36.61 6.19 13.21 0.20

0.038 0.047 0.003 0.008 0.016 0.048 0.009 0.164 < 0.001

3.13

1.21

0.027

-1.89 -3.64 -12.70 -0.26 -3.48 -1.37 1.25 -0.68 -0.94

0.59 1.12 2.50 0.08 1.27 0.50 0.46 0.22 0.41

0.010 0.009 < 0.001 0.007 0.021 0.020 0.020 0.011 0.045

CIE b*

0.55**

AN

0.49*

**

0.59

PT

pH

ED

M

CIE a*

US

CR

IP

-0.24

T

R2a

CE

Anterior

Dependent variable CIE L*

AC

Region

WarnerBratzler shear force

0.76**

CIE a*

0.76**

CIE b*

0.43*

pH

0.59**

Drip loss

0.87**

Phosphoglucomutase 1 Carbonic anhydrase 3 Myosin-1 Heat shock 27 kDa protein Glycerol-3-phosphate dehydrogenase L-lactate dehydrogenase A chain MLC3 Myosin-2 Myosin-1 Heat shock 27 kDa protein Phosphoglucomutase 1 Carbonic anhydrase 3 Myosin-1 L-lactate dehydrogenase A chain Phosphoglucomutase 1 Myosin-2 Myosin-1 Heat shock 27 kDa protein Beta-tropomyosin Phosphoglucomutase 1 Glycerol-3-phosphate dehydrogenase Carbonic anhydrase 3 Myosin-1 Beta-tropomyosin Carbonic anhydrase 3 Heat shock 27 kDa protein L-lactate dehydrogenase A chain MLC2 MLC3 Myosin-1 Carbonic anhydrase 3 Myosin-1 Myosin-1 Glycerol-3-phosphate dehydrogenase L-lactate dehydrogenase A chain Carbonic anhydrase 3

AN

M

0.69**

AC

CIE a*

CE

PT

Posterior CIE L*

0.51**

ED

WarnerBratzler shear force

pH Drip loss

0.40*

0.35* 0.89**

Cooking 0.77* loss Warner0.58** Bratzler Myosin-1 shear force a * Significance of the models: , P < 0.05; ** , P < 0.01.

-0.22 0.40 3.64 0.16

0.10 0.17 0.93 0.07

0.047 0.041 0.003 0.039

-0.22

0.04

< 0.001

-3.75

1.57

0.038

-0.26 2.13 11.95 0.67 15.78 -26.74 -242.94

0.11 0.88 4.38 0.26 6.17 11.76 64.02

0.041 0.035 0.021 0.029 0.029 0.046 0.004

8.27

1.64

0.007

1.99 3.21 25.14 1.57 0.58 0.89

0.55 0.97 4.83 0.38 0.14 0.27

0.023 0.029 0.007 0.014 0.014 0.009

-0.17

0.06

0.013

-1.38 -11.23 0.08 0.48 0.21

0.57 3.47 0.03 0.17 0.07

0.036 0.009 0.029 0.020 0.012

-1.03

0.43

0.038

0.64 -0.07 4.20 -0.90 -6.55 -216.06

0.29 0.03 0.88 0.38 2.52 92.22

0.049 0.043 < 0.001 0.039 0.026 0.041

0.34

0.06

0.005

2.31

0.64

0.022

-1.45

0.60

0.035

-12.56

3.36

0.004

T

0.60**

CR

CIE L*

US

Medial

IP

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights 

The reddish-pink changed to pale-grayish-pink from the anterior to the posterior region. Low water-holding capacity in the medial region was associated to glycolysis

T



IP

enzymes.

High tenderness in the anterior region was closely related with contractile proteins.



Color of the medial region can be distinguished with GAPDH.

AC

CE

PT

ED

M

AN

US

CR



Figure 1

Figure 2

Figure 3

Figure 4