Centrifugal drip is an accessible source for protein indicators of pork

Transcription

MESC-05289; No of Pages 10
Meat Science xxx (2011) xxx–xxx
Contents lists available at ScienceDirect
Meat Science
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i
Centrifugal drip is an accessible source for protein indicators of pork ageing and
water-holding capacity
Alessio Di Luca a, Anne Maria Mullen a,⁎, Giuliano Elia b, Grace Davey c, Ruth M. Hamill a
a
b
c
Teagasc Food Research Centre Ashtown, Dublin 15, Ireland
UCD Conway Institute of Biomolecular and Biomedical Research, Belfield, Dublin 4, Ireland
Functional Genomics & Glycomics Group, Martin Ryan Institute, National University of Ireland Galway, Galway, Ireland
a r t i c l e
i n f o
Article history:
Received 9 July 2010
Received in revised form 23 November 2010
Accepted 20 December 2010
Available online xxxx
Keywords:
Meat quality
Drip loss
Post mortem changes
SDS PAGE
Mass spectrometry
a b s t r a c t
Achieving an improvement in water-holding capacity (WHC) of pork and a reduction in the incidence of pale,
soft and exudative (PSE)- and dark, firm and dry (DFD)-like meat is a major challenge for the swine industry.
Using proteomics, we sought to identify proteins associated with WHC and to monitor postmortem protein
degradation. Twenty longissimus samples were categorised into WHC phenotypes. The centrifugal drip was
subjected to SDS-PAGE and mass-spectrometry. Forty-four proteins were identified in the centrifugal drip
proteome. Changes occurred in volume of five bands across the ageing period, with most significant changes
representing increases between day 3 and day 7. Seven proteins were identified in these bands, most with
functions in glycolysis. One band significantly differed in abundance across WHC phenotypes. Peptide
signatures of the heat shock protein family were identified in this band.
© 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Water-holding capacity (WHC) is the ability of meat to retain
water during cutting, heating, and pressing and can be measured as
drip loss (Hamm, 1960). Incidence of high drip loss represents an
economic problem for the swine industry because drip loss leads to
significant weight loss in raw, cooked and processed meat; losses of
up to 10–12% of carcass weight are observed (Melody, Lonergan,
Rowe, Huiatt, & Huff-Lonergan, 2004; van de Wiel & Zhang, 2007).
Additionally, meat that has had high exudative losses in the form of
drip has a poor appearance and texture. While there are theories on
the intrinsic physico-chemical mechanisms involved in drip formation in meat, e.g. the shrinkage of myofibrils during rigor development
(Offer, 1991) and the occurrence of tissue damage through cutting
(Honikel, 2004), the reasons why drip loss varies from individual to
individual, even in controlled environmental conditions, are not fully
understood (Otto et al., 2007).
As well as being high in drip loss, some samples, termed pale, soft
and exudative (PSE) or PSE-like meat (similar characteristics), display
a combination of attributes which are especially undesirable (Bowker,
Grant, Forrest, & Gerrard, 2000). While in general, a low level of drip
loss is preferred, a subset of pork samples characterised by low drip
loss present an unattractive dark appearance, firm, dry (DFD) and
⁎ Corresponding author. Tel./fax: + 353 1 8059550.
E-mail address: [email protected] (A.M. Mullen).
sticky texture and have a poor shelf life (Newton & Gill, 1981;
Scheffler & Gerrard, 2007). There are a number of theories as to the
causes of high drip loss, PSE and DFD meat. PSE and DFD conditions
are thought to be influenced by a combination of genetics, variability
in energy reserves at time of slaughter, rate of post mortem pH decline
and stress (Fischer, 2007; Newton & Gill, 1981). During the process of
conversion from muscle to meat, a series of biochemical events
contributes to the development of many meat quality traits
(Koohmaraie, 1996; Ouali et al., 2006; Renand, Picard, Touraille,
Berge, & Lepetit, 2001). The influence of the calpain enzyme system
has been highlighted (Koohmaraie & Geesink, 2006; Koohmaraie,
Kent, Shackelford, Veiseth, & Wheeler, 2002; Purintrapiban, Wang, &
Forsberg, 2001) as has the role of programmed cell death (HerreraMendez, Becila, Boudjellal, & Ouali, 2006). However, the impact of
these processes on the physiology of post mortem tissue has not been
completely elucidated.
Thanks to advances in the areas of genomics and proteomics, a
better understanding of the biological processes underpinning meat
quality is now possible (Hollung, Veiseth, Jia, Faergestad, & Hildrum,
2007; Lametsch, 2009). Important steps have already been made in
understanding the post mortem processes responsible for conversion
of muscle to meat, e.g. proteomic studies identifying the importance
of protein degradation (Lametsch, 2009; Lametsch & Bendixen, 2001;
Lametsch, Roepstorff, & Bendixen, 2002). The proteomic approach has
also been applied to study muscles divergent in WHC traits, such as
PSE meat compared with normal meat (Laville et al., 2005), and high
versus low drip loss (van de Wiel & Zhang, 2007). However, including
a diversity of WHC phenotypes in a single study would help us to
0309-1740/$ – see front matter © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2010.12.033
Please cite this article as: Di Luca, A., et al., Centrifugal drip is an accessible source for protein indicators of pork ageing and water-holding
capacity, Meat Science (2011), doi:10.1016/j.meatsci.2010.12.033
2
A. Di Luca et al. / Meat Science xxx (2011) xxx–xxx
develop a more complete understanding of the regulation of waterholding capacity. The aim of this study was to examine exudate
collected from pork longissimus to identify proteins that correlate with
WHC and with post mortem ageing.
2. Materials and methods
2.1. Animal sampling
Thirty one Large White × Landrace/Large White gilts were electrically stunned and slaughtered under controlled conditions in a pilot
abattoir at Teagasc, Food Research Centre, Ashtown. Tissue samples
were collected from the longissimus thoracis et lumborum (LTL) muscle
at 1 day, 3 days and 7 days post mortem. After slaughter the carcass
was stored at 4 °C until day 1 post mortem, then de-boned. The entire
LTL muscle was removed from the carcass. 2.54 cm subsamples were
taken on days 1, 3, and 7, for meat quality analysis with loins being
vacuum packaged and stored at 4 °C over this period.
2.1.1. Centrifugal drip collection
Exudates were collected from muscle tissue following a modified
protocol of Bouton, Harris, and Shorthose (1971). We reduced the
centrifugation speed to 5911 ×g at 4 °C and increased the amount of
samples to three 8 g cores (12 mm diameter × 2.5 cm) taken from
slices of LTL muscle from each sample. Care was taken to avoid
obvious pieces of fat. These were centrifuged in polyalcomer
centrifuge tubes (25 × 89 m; Beckman) for 60 min (Beckman
Optima™ XL — 100K Ultracentrifuge, USA). After centrifugation,
the exudate was snap frozen in liquid nitrogen and stored at
−80 °C until required. The amount of exudate collected using this
technique ranged from about 80 μl to 2 ml (average of about
500 μl). The concentration of the centrifugal drip samples from all
31 animals (at days 1, 3 and 7 post mortem) was determined using
the Bio-Rad Protein Assay Kit (Bio-Rad Labs, Hercules, CA, USA),
following the Bradford method and were found to be between 90
and 160 μg/μl (Bradford, 1976). Bovine serum albumin was included
as a reference.
2.2.3. Drip loss
Drip loss was determined according to the bag method of Honikel
(1998). Samples about 2.5 cm thick and weighing approximately 80 g
were removed from the LTL of each animal. They were suspended by
string in an expanded bag, so that the meat did not come in contact
with the bag. The samples were then suspended at 4 °C for 48 h, at
which time the surface was lightly blotted with a paper tissue and reweighed. Drip loss was then expressed as a percentage of the original
weight of the steak.
2.2.4. Warner Bratzler shear force (WBSF)
WBSF was measured on cooked meat with (2.54 cm thick) per
muscle according to the protocol of Wheeler, Shackelford, and
Koohmaraie (1996). A transversal section of the LTL muscle for each
animals on day 1, day 3 and day 7 was cooked to a core temperature of
75 °C (Minitherm HI8751 temperature meter and probe, Hanna
Instruments Ltd., Bedfordshire, UK) in a water bath 77 °C (model
Y38, Grant Instruments Ltd., Barrington, UK), tempered at room
temperature and left to cool at 4 °C overnight. Cores (1.25 cm, parallel
to longitudinal orientation of fibres) were then taken at random
within the steak. Steaks were cored straight from the fridge and when
cores reached room temperature they were sheared using the Instron
model 5543 and Bluehill software, Instron Ltd. (Buckinghamshire,
UK).
2.2.5. WHC phenotype categorisation
Animals were categorised into meat quality types for further study
based mainly on drip loss and pH values as described below. Drip loss
data (drip loss N 6%) together with pH at 45 min (pH45 b 6.2) postexsanguination and Hunter L* measurements were used to detect
signs of PSE. Drip loss (drip loss b 2.2%), ultimate pH (pHu N 5.6) and L*
colour measurements were used to detect signs of DFD. Animals not
displaying signs of PSE or DFD meat, but showing respectively high
drip loss (drip loss N 5%, pH45 N 6.17) (HDrip) and low drip loss (drip
loss b 2.9%, pHu b 5.56) (LDrip) were selected to specifically examine
divergence in drip loss. Samples with drip loss between 3.5 and 4.4%,
pH45 N 6.2 and pHu b 5.8 were selected as being representative of
intermediate phenotype (IP).
2.2. Meat quality measurements
2.3. Proteomic analysis
A variety of technological quality measurements were performed
on loins removed from the left side of each carcass over a 7 day period
post slaughter.
2.2.1. pH and temperature
Loin pH and temperature were measured at 45 min, 2, 3, 4, 5, 6 and
24 h post slaughter. pH was measured using a portable pH meter
(Orion Research Inc., Boston, MA 02129, USA) and an Amagruss pH
electrode (pH/mV Sensors Ltd., Murrisk-Westport, Co. Mayo, Ireland),
which was adjusted for muscle temperature before being inserted
approximately 6 cm into muscles. Insertion point on the LTL was
between the 10th and 11th rib. Before analysis the pH meter was
calibrated using standard phosphate buffers (pH 4.01 and 7.00,
Radiometer, Copenhagen, Denmark). The electrode was rinsed
thoroughly with distilled water between measurements.
2.2.2. Colour
A transverse section of the LTL muscle was used to measure colour
after 3 h blooming at 1, 3 and 7 days post slaughter using MiniScan XE
Plus (Hunter Associates Laboratory, Inc., Reston, USA), with a D65
illuminant, 10° standard observer angle and 32 mm aperture size. The
instrument was calibrated before each series of measurements using a
white tile (L* = 100) and black glass (L* = 0). For all colour
measurements, triplicate readings were averaged for each sample
and L* (lightness), a* (redness) and b* (yellowness) values were
determined using the 1976 CIELAB system.
2.3.1. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
For this preliminary investigation of the centrifugal drip proteome,
SDS-PAGE was selected in preference to 2D SDS-PAGE. The intermediate samples at three time-points and all 5 phenotypic categories
were analysed by SDS PAGE. Technical duplicates of four biological
replicates for each of the phenotypes and time-points were visually
assessed for reproducibility (93 lanes in total). Moreover, a mass
spectrometry analysis of a single gel lane dissected into 24 strips was
carried out permitting first profiling of the proteins present in the
centrifugal drip substrate.
Fifteen μg of protein from each sample were subjected to
electrophoresis using 10% polyacrylamide resolving gels with a 4%
stacking gel (Laemmli, 1970). Protein samples were denatured by
mixing with sample buffer (2% SDS, 10% glycerol, 0.1 M Tris–HCl at pH
6.8, 1% β-mercaptoethanol and traces of bromophenol blue) and
heated at 95 °C for 5 min. A wide range molecular weight standard
(Sigma) was run on each gel to determine protein band molecular
weights. Gels (10.1 × 8.2 cm) were run in a Mini-PROTEAN Tetra Cell
system (Bio-Rad, USA). The gels were run at constant 100 V for about
110 min. Gels were stained with Coomassie Blue (0.01% Coomassie
Blue R-250, 30% methanol, 10% acetic acid) overnight and then
destained (10% methanol, 10% acetic acid) for about 2 h. The gels were
then scanned using a densitometric scanner (GS-800 Bio-Rad) and
band volume for target lanes was determined using Quantity one
4.5.2. software (Bio-Rad) in order to identify if there were significant
3
Table 1
Mean ± standard error of meat quality traits relevant to WHC in the Large White × Landrace/Large White population (LTL, longissimus thoracis et lumborum; HDrip, high drip loss;
LDrip, low drip loss; IP, intermediate phenotype; PSE, pale, soft and exudative; DFD = dark, firm and dry); L*, lightness; a*, redness; b*, yellowness; WBSF, Warner-Bratzler shear
force; N, Newtons.
Trait
Overall dataset (n = 31)
PSE samples (n = 4)
HDrip samples (n = 4)
IP samples (n = 4)
LDrip samples (n = 4)
DFD samples (n = 4)
pH 45 min
pH 3 h
pH 24 h
CIE L⁎ (24 h)
CIE a⁎ (24 h)
CIE b⁎ (24 h)
Drip loss (%)
WBSF day 1 (N)
WBSF day 3 (N)
WBSF day 7 (N)
6.34 ± 0.27
5.85 ± 0.28
5.55 ± 0.14
54.93 ± 3.55
7.21 ± 1.46
15.14 ± 0.86
4.00 ± 2.11
43.8 ± 6.71
44.33 ± 7.67
33.45 ± 4.86
5.96 ± 0.26
5.64 ± 0.11
5.42 ± 0.06
58.74 ± 5.07
8.47 ± 0.84
16.14 ± 0.86
7.76 ± 1.31
44.00 ± 4.35
42.8 ± 10.3
33 ± 6.93
6.35 ± 0.12
5.86 ± 0.28
5.62 ± 0.08
54.80 ± 3.25
7.97 ± 2.46
15.48 ± 0.65
6.1 ± 1.45
46.15 ± 7.6
52.81 ± 10.69
37.91 ± 6.42
6.43 ± 0.2
5.89 ± 0.22
5.55 ± 0.13
55.42 ± 2.01
7.32 ± 0.67
15.43 ± 0.48
3.91 ± 0.38
45.67 ± 3.16
40.31 ± 5.2
32.01 ± 3.5
6.49 ± 0.20
5.95 ± 0.40
5.49 ± 0.06
53.77 ± 4.05
7.67 ± 1.69
15.35 ± 0.74
2.54 ± 0.3
40.2 ± 4.6
43.47 ± 7.16
31.78 ± 1.22
6.41 ± 0.12
5.94 ± 0.24
5.71 ± 0.11
52.26 ± 0.8
6.38 ± 1.2
14.36 ± 0.76
1.91 ± 0.14
43.00 ± 11.34
42.17 ± 3.83
31.97 ± 6.17
2.3.2. Statistical analysis
To test for an association of protein abundance with WHC, the
volume of gel bands for each biological replicate was analysed across
WHC conditions (PSE, DFD, HDrip, LDrip, and IP) using the GLM
procedure in SAS v.9.1 (SAS Institute, Carry, NC, USA). Condition was
included as a fixed effect. Where a significant association was
observed, Tukey–Kramer post hoc analysis was applied to determine
which phenotype categories differed significantly.
In order to examine the impact of ageing on the drip proteome,
band volumes of intermediate phenotype samples were modelled
using a repeated measures ANOVA procedure in SAS v.9.1. Time-point
was included in each model as a fixed effect and animal as a random
effect. Each band was analysed in a separate model. For significant
bands, Tukey–Kramer post hoc analysis at the 0.05 level was applied to
contrast time-points.
Statistical analyses were used to associate individual protein/
fragment bands from pork centrifugal drip samples, with differences
across phenotypes and days post mortem.
2.3.3. Mass spectrometry (MS) analysis
In order to identify the protein species present in the centrifugal
drip fraction, a single sample gel lane (Fig. 5) was removed and
dissected into 24 strips, each strip was washed in 200 mM NH4HCO3, in
200 mM NH4HCO3/ACN 2:3, in 50 mM NH4HCO3 and ACN, and then
dried by vacuum centrifugation. Trypsin (Sequencing Grade Modified,
Promega) was dissolved in 50 mM NH4HCO3, then added to each
sample and incubated at 37 °C, with shaking, overnight. Peptides were
extracted with two different concentrations of ACN/0.2% TFA (30% and
70%). The extracted peptides were concentrated in a speed vac at 45 °C
to dry. All samples were run on a Finnigan LTQ (USA) mass
spectrometer connected to a Surveyor chromatography system
incorporating an auto-sampler. Tryptic peptides were resuspended in
0.1% formic acid and were separated by means of a modular Cap LC
system (Finnigan) connected directly to the source of the LTQ. Each
sample was loaded onto a Biobasic C18 Picofrit™ column (100 mm
length, 75 μm ID) at a flow rate of 30 nl min− 1. The samples were then
eluted from the C18 Picofrit™ column by an increasing acetonitrile
gradient. The mass spectrometer was operated in positive ion mode
with a capillary temperature of 200 °C, a capillary voltage of 46 V, a tube
lens voltage of 140 V and with a potential of 1800 V applied to the frit.
All data were acquired with the mass spectrometer operating in
automatic data dependent switching mode. An MS scan was performed
to select the 5 most intense ions prior to MS/MS analysis. MS/MS
spectra were sequence database searched using TurboSEQUEST
against Uniprot/Swissprot database (Jan 2008). The following search
parameters were used: precursor-ion mass tolerance of 1.5 Da,
fragment ion tolerance of 1.0 Da with methionine oxidation and
cysteine carboxyamidomethylation specified as differential modifications and a maximum of 2 missed cleavage sites allowed. The criterion
of sufficient supporting evidence for protein identification was a
minimum of 2 peptides.
3. Results
3.1. Identification of individuals divergent in meat quality attributes
Table 1 shows the mean and the standard errors of key meat quality
parameters in the Large White × Landrace/Large White population. Drip
loss varied approximately 10 fold amongst the 31 animals with the
lowest recorded values at 0.9% and the highest at 9.5% (mean 4.0 ± 2.1
std). From this panel 20 samples were categorised as representative of
five divergent meat quality types (as described in 2.2.5) and were used
for proteomic analysis. Drip loss % was significantly associated with
phenotype category (pb 0.001). Shear force values across phenotype
categories may be seen in Table 1. One-way ANOVA revealed that shear
force was not associated with phenotype. Warner Bratzler shear force
values for the 4 intermediate phenotype (IP) samples across three
timepoints postmortem are presented in Fig. 1. Mean shear force
declined across the ageing period from a mean of 45.67 N at day 1 to
32.01 N at day 7.
3.2. SDS PAGE
Fig. 2 shows 8 representative lanes from a 10% SDS-PAGE of the
centrifugal drip sample. Three gel lanes are from the comparison of
the intermediate phenotype (IP) at days 1, 3 and 7 post mortem, and 5
gel lanes are from the comparison of phenotypes at day 1 post mortem
(PSE, DFD, IP, HDrip, and LDrip). The molecular weight standard is
present in the centre of the gel. Seventeen protein bands across all
60.00
50.00
WBSF (N)
differences in band volume across phenotypes and the post mortem
period.
40.00
30.00
20.00
10.00
0.00
Day 1
Day 3
Day 7
Days post mortem
Fig. 1. Means and standard deviation of Warner Bratzler Shear Force (WBSF) values for
4 IP samples monitored across the three days post mortem.
4
Fig. 2. Synthetic SDS PAGE gel of the centrifugal drip samples showing example lanes for intermediate phenotypes (IP) at day 1, day 3 and day 7 post mortem, and divergent
phenotypes at day 1 post mortem (pale, soft exudative (PSE), dark, firm dry (DFD), high drip loss (HDrip), low drip loss (LDrip)). The molecular weight standard is indicated by MW.
Numbers (1 to 17) at the side of the image show the location of the 17 bands detected by the densitometer. Arrows indicate the bands that are significantly changing in at least one
comparison following statistical analysis.
Band volume
samples were detected by the software Quantity one 4.5.2. (Bio-Rad)
(Fig. 2).
Least square means and standard errors for band 5 (MW ~68 kDa)
volume are shown in Fig. 3. ANOVA showed that this band was
significantly associated (df = 4, F = 3.36, p = 0.038) with WHC
phenotype. The mean band volume was significantly higher in LDrip
than in PSE-like samples at the 0.05 level (Fig. 3). Other comparisons
were not significant. Muscle of HDrip meat showed volumes similar to
PSE, whereas the volumes of DFD phenotype samples were similar to
LDrip. Further, the abundance of proteins in this band for samples
with intermediate drip loss levels (IP) showed intermediate intensities compared to high and low drip loss samples, though band
volumes were more similar to low drip rather than high drip samples
(Fig. 3). There was a significant correlation between band 5 volume
and drip loss level, irrespective of phenotypic category (r2 = 0.295,
p = 0.013). This was similar to the correlation between drip loss and
pH at 45 min (r2 = 0.32, p = 0.001), whereas there were no correlations between drip loss and pH at 2 h, 3 h and 24 h time points.
ANOVA also showed that the volume of five bands was
significantly changing across days post mortem (days 1, 3 and 7) in
the four intermediate phenotype (IP) samples (Fig. 4). These were
bands 4 (~83 kDa) (df = 2, F = 14.08, p = 0.005), band 5 (~ 68 kDa)
(df = 2, F = 9.33, p = 0.014), band 6 (~63 kDa) (df = 2, F = 18.44,
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
b
ab
ab
ab
a
PSE
DFD
IP
HDrip
LDrip
Phenotypes
Fig. 3. Mean and standard error of volume of band 5 (Fig. 2) across five phenotypes
(PSE, DFD, LDrip, HDrip and IP) at day 1 post mortem. Different superscripts indicate
significantly different band volumes.
Fig. 4. Plots of significantly changing band volumes across the three days (days 1, 3 and
7) post mortem in the four animals with intermediate drip loss phenotype. Least square
means and standard errors of the three lower abundance bands are displayed in Panel A
and of the two higher abundance bands are displayed in Panel B.
5
Of the five bands that were significantly different in the two
comparisons, band 4 was located in strip E, band 5 in strip F, band 6 in
strip G, band 10 in strip L, and band 13 in strip O (Table 2). Band 5 was
significantly different according to phenotype and time post mortem.
Mass spectrometry and bioinformatics analysis indicate the presence
of heat shock protein 70 (HSP 70) in this strip. Four peptides were
identified for this protein (R.TTPSYVAFTDTER.L; R.STAGDTHLGGEDFDNR.L; K.NSLESYAFNMK.S; K.DAGTIAGLNVLR.I). However, as
some of these peptides are shared with other heat shock protein
family members (Heat shock cognate 70 kDa, Swiss-Prot P08108;
Heat shock protein 68, Swiss-Prot O97125; 78 kDa glucose-regulated
protein homolog, Swiss- Prot P36604), the possibility, that some other
HSP may also be present in band 5 cannot be ruled out. HSP 70 is
involved in the biological process of stress response and protein
folding.
In addition to band 5, four other bands significantly changed in
intensity across the days post mortem. Peptides mapping to 6phosphofructokinase were identified in band 4, serum albumin
precursor in band 6, enolase, beta-enolase, alpha-enolase and creatine
kinase M-type in band 10, creatine kinase M-type, phosphoglycerate
kinase 1, fructose-bisphosphate aldolase A, and glyceraldehyde-3phosphate dehydrogenase in band 13. The full list of proteins/
fragment identified is shown in Table 2 together with their biological
function obtained using PANTHER analysis (Thomas et al., 2006). All
identified proteins, with the exception of serum albumin and creatine
kinase, are metabolic enzymes with functions in glycolysis.
4. Discussion
Fig. 5. Gel lane indicating where the strips were dissected and subsequently digested
with trypsin prior to mass spectrometric analysis.
p = 0.003), band 10 (~42 kDa) (df = 2, F = 5.16, p = 0.05) and band 13
(~31 kDa) (df = 2, F = 6.01, p = 0.037), although for bands 10 and 13,
the overall model was not significant. For band 6, there was also a
significant effect of animal on band volume (df = 3, F = 11.77,
p = 0.006), suggesting that this band is highly labile. There was a
significant difference (p = 0.05) between band volumes at days 1 and
7 and between volumes at days 3 and 7, with band volumes at days 1
and 3 being similar (Fig. 4). Bands 5, 6, 10, and 13 showed band
volume profiles that were similar to each other over the days post
mortem, with lowest volumes at day 3 and highest volumes at day 7.
The volume at days 3 and 7 were significantly different for each of
these bands (Fig. 4). Band 4 showed an increase in volume across all
time-points post mortem.
3.3. Mass spectrometric analysis
The 17 protein/fragment bands detected by the densitometric
analysis are identified in the figures and in the mass spectrometry
tables by numbers 1–17 (Fig. 2 and Table 2). One hundred and six
different proteins/fragments were identified by mass spectrometry
and bioinformatic analysis among 24 dissected and digested strips
from the single gel lane. Table 2 shows the protein sequences
identified using the criteria described above. Forty four proteins were
identified, with many (possibly fragments) of these present in more
than one gel strip.
We found that centrifugal drip is rich in protein and the SDS PAGE
band profile shows similarity to gels on which sarcoplasmic extracts
were run e.g. Fig. 4 in Okumura, Yamada, and Nishimura (2003); see
also Savage, Warriss, and Jolley (1990). However, the centrifugal drip
is obtained via a more straightforward extraction method. The
majority of the proteins identified were muscle isoforms of enzymes
involved in glucose metabolism (Table 2). SDS PAGE gels indicated
that the centrifugal drip proteome is reproducible across individuals
and post mortem time-points (93 samples at three time-points plus
replicates were run by SDS PAGE, data not shown). In contrast to the
myofibrillar fraction (Okumura et al., 2003; Zapata, Zerby, & Wick,
2009), the drip was not predominated by highly abundant proteins
such as actin, titin, nebulin and myosin, which constitute about 80% of
total muscle proteins (Lametsch et al., 2006). This makes it possible to
study less expressed proteins which may influence or be markers of
phenotype. Many of the proteins/peptides identified were detected in
more than one band, e.g. creatine kinase was observed in 4 bands
(Purintrapiban et al., 2001), and thus probably represent multiple
degradation products.
In this study, the volume of five protein bands was observed to
increase during the post mortem ageing period, with the biggest
differences observed between days 3 and 7 post mortem. During the
post mortem conversion of muscle to meat, many meat quality traits
are developed (Ouali et al., 2006). The main biochemical processes
(e.g. proteolytic tenderisation) that occur during this conversion
have been elucidated (Koohmaraie, 1996; Koohmaraie & Geesink,
2006; Quali, 1992; Scheffler & Gerrard, 2007). However, the
molecular mechanisms underpinning the post mortem changes are
still not completely clear (Ouali et al., 2006). Six proteins, most with
functions in glycolysis, e.g. 6-phosphofructokinase in band 4
(Rhoades et al., 2005), were identified in the 5 changing bands.
Through post mortem ageing, some intact proteins will be degraded
and decline in abundance with muscle ageing, whereas degradation
products will increase in abundance, and possibly also disappear
again as they are further degraded. The largest changes occurred
between day 3 and day 7 and a number of bands were observed to
reduce in abundance between days 1 and 3 and then increase
6
Table 2
Protein fragments in porcine centrifugal drip identified by mean of ion trap mass spectrometer. aStrips that were digested and analysed by MS. bBand detected by densitometer, if
any. cAccession number in the UniProt database. dMolecular weight of the protein. eTurboSEQUEST score. ⁎Methionine oxidation. #Carboxyamidomethylation.
Stripa
Bandb
Swiss-Protc
Identification
Match peptide
MW (kDA)d
D
3
Q3B7M9
Glycogen phosphorylase, brain form (glycogen metabolism)
96.3
48.23
D
3
P79334
Glycogen phosphorylase, muscle form (glycogen metabolism)
97.2
100.22
D
3
Q7VR21
Acyl carrier protein (phosphate metabolism)
86.9
16.14
E
4
Q0IIG5
6-Phosphofructokinase, muscle type (glycolysis)
85.2
20.18
F
5
A5A8V7
Heat shock 70 kDa protein 1-like (immune system process; protein
metabolic process; response to stress)
70.3
40.76
G
6
P08835
Serum albumin precursor (transport)
69.6
40.24
H
7
P36871
Phosphoglucomutase-1 (monosaccharide metabolism)
61.4
106.28
H
7
P00548
Pyruvate kinase muscle isozyme (glycolysis)
I
8
P00548
I
8
P11979
Pyruvate kinase isozyme M1 (glycolysis)
I
8
P11974
Pyruvate kinase isozymes M1/M2 (glycolysis)
J
NO
P00548
J
NO
P11979
Pyruvate kinase isozyme M1 (glycolysis)
J
NO
P08059
Glucose-6-phosphate isomerase (glycolysis, gluconeogenesis)
K
9
P13549
Elongation factor 1-alpha, somatic form (protein biosynthesis)
R.NLAENISR.V
K.TC*AYTNHTVLPEALER.W
R.YEFGIFNQK.I
K.VIFLENYR.V
R.VLYPNDNFFEGK.E
R.TQQHYYEKDPKR.I
R.TQQHYYEKDPK.R
R.NNVVNTMR.L
R.VSALYKNPR.E
R.VEDVERLDQK.G
R.M#SLVEEGAVKR.I
R.M#SLVEEGAVK.R
R.MSLVEEGAVKR.I
K.APNDFNLK.D
R.MSLVEEGAVK.R
-.M#SIVEEKVK.T
-.MSIVEEKVK.T
R.IGLIQGNR.M
R.LPLM#EC*VQVTK.D
R.TTPSYVAFTDTER.L
R.STAGDTHLGGEDFDNR.L
K.NSLESYAFNM#K.S
K.DAGTIAGLNVLR.I
K.HKPHATEEQLR.T
R.DTYKSEIAHR.F
K.YIC*ENQDTISTK.L
K.ADFTEISK.I
R.SM#PTSGALDR.V
K.TQAYQDQKPGTSGLR.K
R.IAAANGIGR.L
R.LSGTGSAGATIR.L
R.QEATLVVGGDGR.F
R.YDYEEVEAEGANK.M
K.FNISNGGPAPEAITDK.I
K.VDLGVLGK.Q
K.FFGNLM#DASK.L
K.IALYETPTGWK.F
K.FFGNLMDASK.L
K.GSGTAEVELKK.G
R.APIIAVTR.N
R.GDLGIEIPAEK.V
K.IENHEGVR.R
R.EAEAAM#FHR.Q
K.GSGTAEVELKK.G
K.GSGTAEVELK.K
R.EAEAAMFHR.Q
R.APIIAVTR.N
K.GDYPLEAVR.M
R.NTGIIC*TIGPASR.S
R.AGKPIIC*ATQM#LESM#IK.K
K.KGVNLPGAAVDLPAVSEK.D
K.GVNLPGAAVDLPAVSEK.D
R.GDLGIEIPAEK.V
R.VNLAM#NVGK.A
K.ITLDNAYM#EK.C
R.VNLAMNVGK.A
K.ITLDNAYMEK.C
R.LDIDSPPITAR.N
K.DPVQEAWAEDVDLR.V
R.LNFSHGTHEYHAETIK.N
K.QKGPDFLVTEVENGGFLGSK.K
K.GPDFLVTEVENGGFLGSK.K
K.GSGTAEVELK.K
R.APIIAVTR.N
K.GDYPLEAVR.M
R.NTGIIC*TIGPASR.S
R.GDLGIEIPAEK.V
R.VNLAM#NVGK.A
R.LNFSHGTHEYHAETIK.N
R.LDIDSPPITAR.N
R.LFEGDKDR.F
R.RLFEGDKDR.F
K.STTTGHLIYK.C
K.IGGIGTVPVGR.V
58
58
Scoree
30.16
120.3
58
68.24
58
20.20
58
50.23
58
30.23
63.1
20.17
50.2
18.18
7
Table 2 (continued)
Stripa
Bandb
Swiss-Protc
Identification
Match peptide
MW (kDA)d
Scoree
K
9
P00548
58
30.16
K
9
Q3ZC09
Beta-enolase (glycolysis)
47.1
30.20
L
10
Q1KYT0
Beta-enolase (glycolysis)
47
18.29
L
10
P17182
Alpha-enolase (glycolysis)
47.1
36.28
L
10
Q9XSC6
Creatine kinase M-type (muscle contraction)
43
20.28
L
10
Q27877
Enolase (glycolysis)
47
18.24
M
11
Q9XSC6
43
156.33
M
11
Q3T0P6
Phosphoglycerate kinase 1 (glycolysis)
44.5
108.32
M
11
P04075
Fructose-bisphosphate aldolase A (glycolysis)
39.4
34.25
M
11
P06733
Alpha-enolase (glycolysis)
47.1
14.22
N
12
P09972
Fructose-bisphosphate aldolase C (glycolysis)
39.4
20.35
N
12
P04075
Fructose-bisphosphate aldolase A (glycolysis)
39.4
170.38
N
12
Q9XSC6
N
12
P53447
Fructose-bisphosphate aldolase B (glycolysis)
K.GSGTAEVELKK.G
K.GDYPLEAVR.M
R.GDLGIEIPAEK.V
K.TAIQAAGYPDK.V
R.GNPTVEVDLHTAK.G
R.NGKYDLDFK.S
K.TLGPALLEK.K
R.NGKYDLDFKSPDDPSR.H
K.NYPVVSIEDPFDQDDWK.T
K.LAM#QEFM#ILPVGASSFR.E
K.LAMQEFMILPVGASSFR.E
K.LAM#QEFMILPVGASSFR.E
K.LAMQEFM#ILPVGASSFR.E
R.GTGGVDTAAVGSVFDVSNADR.L
K.DLFDPIIQDR.H
K.LAQDSGWGVM#VSHR.S
K.LAQDSGWGVMVSHR.S
K.TDLNHENLK.G
K.GQSIDDM#IPAQK
K.AEEEYPDLSK.H
K.HKTDLNHENLKGGDDLDPNYVLSSR.V
K.GQSIDDMIPAQK
K.ALTLEIYKK.L
K.LNFKAEEEYPDLSK.H
K.GGDDLDPNYVLSSR.V
K.TDLNHENLKGGDDLDPNYVLSSR.V
K.RGTGGVDTAAVGSVFDVSNADR.L
K.DLFDPIIQDR.H
K.LSVEALNSLTGEFK.G
R.LGSSEVEQVQLVVDGVKLM#VEM#EK.K
K.SFLVWVNEEDHLR.V
R.LGSSEVEQVQLVVDGVK.L
R.FHVEEEGKGK.D
R.VDFNVPM#KNNQITNNQR.I
K.LTLDKLDVK.G
K.VSHVSTGGGASLELLEGK.V
K.LGDVYVNDAFGTAHR.A
K.WNTEDKVSHVSTGGGASLELLEGK.V
K.VLNNM#EIGTSLFDEEGSK.I
K.VLNNMEIGTSLFDEEGSK.I
K.ITLPVDFVTADKFDENAK.T
K.AC*ADPAAGSVILLENLR.F
K.ALESPERPFLAILGGAK.V
K.RLQSIGTENTEENRR.F
R.LQSIGTENTEENRR.F
K.GILAADESTGSIAK.R
K.VDKGVVPLAGTNGETTTQGLDGLSER.C
R.HIADLAGNSEVILPVPAFNVINGGSHAGNK.L
K.DATNVGDEGGFAPNILENKEGLELLK.T
K.VDKGVVPLAGTDGETTTQGLDGLSER.C
K.GVVPLAGTDGETTTQGLDGLSER.C
K.AAQEEYVKR.A
K.AAQEEYVK.R
K.RALANSLAC*QGK.Y
R.LQSIGTENTEENR.R
R.ALANSLAC*QGK.Y
K.GILAADESTGSIAKR.L
R.QLLLTADDR.V
R.I]VAPGKGILAADESTGSIAK.R
K.VDKGVVPLAGTNGETTTQGLDGLSER.C
K.IGEHTPSALAIM#ENANVLAR.Y
R.YASIC*QQNGIVPIVEPEILPDGDHDLK.R
R.VNPC*IGGVILFHETLYQK.A
R.QLLLTADDRVNPC*IGGVILFHETLYQK.A
K.C*PLLKPWALTFSYGR.A
R.YASIC*QQNGIVPIVEPEILPDGDHDLKR.C
K.GVVPLAGTNGETTTQGLDGLSER.C
K.ADDGRPFPQVIK.S
K.GILAADESTGSIAK.R
K.GGDDLDPNYVLSSR.V
R.LGSSEVEQVQLVVDGVKLM#VEM#EK.K
R.LGSSEVEQVQLVVDGVK.L
K.DLFDPIIQDR.H
R.YASIC*QMNGLVPIVEPEILPDGDHDLQR.C
43
48.29
39.6
18.26
(continued on next page)
8
Table 2 (continued)
Stripa
Bandb
Swiss-Protc
Identification
Match peptide
O
13
P10096
Glyceraldehyde-3-phosphate dehydrogenase (glycolysis)
O
13
P80534
Glyceraldehyde-3-phosphate dehydrogenase, muscle (glycolysis)
O
13
Q2KJE5
Glyceraldehyde-3-phosphate dehydrogenase, testis-specific (glycolysis)
P
NO
P10096
P
NO
P00339
L-lactate
Q
NO
P10096
Q
NO
Q9XSC6
Q
NO
Q2KJE5
Q
NO
P00339
L-lactate
R
14
Q2KJE5
S
15
Q32KV0
Phosphoglycerate mutase 2 (glycolysis)
S
15
Q3SZ62
Phosphoglycerate mutase 1 (glycolysis)
S
15
Q0BBK5
2,3-Bisphosphoglycerate-dependent phosphoglycerate mutase
S
15
Q3SZX4
Carbonic anhydrase 3
T
NO
Q5E956
Triosephosphate isomerase (glycolysis, gluconeogenesis, fatty acid)
U
16
P00570
Adenylate kinase isoenzyme 1 (nucleotide and nucleic acid metabolism)
dehydrogenase A chain (glycolysis)
dehydrogenase A chain (glycolysis)
between days 3 and 7. As the shear force also decreased across the
ageing period (Fig. 1), this adds to the evidence that technological
properties of meat are likely to be correlated to the underlying
proteomic processes such as degradation of myofibrillar and
sarcoplasmic proteins (Hopkins & Thompson, 2002; Koohmaraie,
1996; Lametsch et al., 2003). Considerable modulation of the muscle
proteome in the later stages of ageing has been shown elsewhere.
For example during cold storage of beef a drop in content of titin,
desmin and Tn-T was observed, simultaneously with the increase in
their degradation products on day 10 (Iwanowska et al., 2010). The
faintest of the five bands, Band 4 in which only one protein was
R.YASIC*QM#NGLVPIVEPEILPDGDHDLQR.C
R.Y]ASICQM#NGLVPIVEPEILPDGDHDLQR.C
K.RVIISAPSADAPM#FVM#GVNHEK.Y
R.VIISAPSADAPM#FVM#GVNHEK.Y
K.RVIISAPSADAPM#FVMGVNHEK.Y
K.RVIISAPSADAPMFVM#GVNHEK.Y
R.VPTPNVSVVDLTC*RLEKPAK.Y
R.VIISAPSADAPM#FVMGVNHEK.Y
R.VIISAPSADAPMFVM#GVNHEK.Y
K.AITIFQERDPANIK.W
K.RVIISAPSADAPMFVMGVNHEK.Y
R.VIISAPSADAPMFVMGVNHEK.Y
K.VIHDHFGIVEGLM#TTVHAITATQK.T
K.VIHDHFGIVEGLMTTVHAITATQK.T
R.VVDLM#VHM#ASK.E
R.VVDLM#VHM#ASKE
R.VVDLMVHM#ASKE
R.VVDLM#VHMASKE
K.IVSNASC*TTNC*LAPLAK.V
R.VVDLMVHMASK.E
R.VVDLMVHMASKE
K.LTGMAFRVPTPDVSVVDLTC*R.L
R.VPTPDVSVVDLTC*R.L
R.VIISAPSADAPMFVMGVNHEK.Y
K.VIHDHFGIVEGLM#TTVHAITATQK.T
K.VTLTPEEEAHLKK.S
K.NLHPELGTDADKEHWK.A
K.VTLTPEEEAHLK.K
K.DQLIHNLLKEEHVPHNK.I
K.ELADEIALVDVM#EDK.L
K.AITIFQERDPANIK.W
K.GQSIDDM#IPAQK
R.VPTPDVSVVDLTC*R.L
K.VTLTPEEEAHLKK.S
K.NLHPELGTDADKEHWK.A
K.DQLIHNLLKEEHVPHNK.I
R.VPTPDVSVVDLTC*R.L
R.HGESTWNQENR.F
R.FLGDEETVRK.A
R.SFDIPPPPM#DEK.H
R.FLGDEETVR.K
R.KAMEAVAAQGK.A
K.AMEAVAAQGK.A
R.VLIAAHGNSLR.G
R.HYGALSGLNK.A
R.HYGALSGLNKAETAAK.F
R.VVFDDTYDR.S
R.NWRPPQPIK.G
K.TATPQQAQEVHEK.L
K.LDEREAGITEK.V
K.IAVAAQNC*YK.V
R.IIYGGSVTGATC*K.E
R.HVFGESDELIGQK.V
K.VANGAFTGEISPGM#IK.D
R.GETSGRVDDNEETIKK.R
R.GKM#LSEIM#EK.G
MW (kDA)d
Scoree
35.8
116.41
39.4
60.20
43.3
18.23
35.8
30.38
36.6
50.27
35.8
20.39
43
20.29
43.3
18.25
36.6
30.23
43.3
20.17
28.7
38.23
28.9
24.20
27.9
14.21
29.3
18.16
26.7
60.24
21.6
20.21
identified (6-phosphofructokinase fragment), showed an increase in
intensity between days 1 and 3 and also between day 3 and day 7,
suggesting that higher resolution separative methods could permit
more accurate profiling of individual proteins/fragments across the
ageing period.
Peptides mapping to 6-phosphofructokinase were identified in
band 4, serum albumin precursor in band 6, enolase, beta-enolase,
alpha-enolase and creatine kinase M-type in band 10, and glyceraldehyde-3-phosphate dehydrogenase in band 13. The ~43 kDA band
(band 10) contained a number of glycolytic enzyme fragments
(enolase, beta-enolase, alpha-enolase and creatine kinase M-type).
Several of these proteins are known targets for the calpain proteolytic
system (Houbak, Ertbjerg, & Therkildsen, 2008; Lametsch et al., 2002;
Purintrapiban et al., 2001) and this enzyme together with other
endogenous enzymatic systems is believed to play an important role
in the process of post mortem proteolysis and meat tenderisation
during the post mortem ageing process (Herrera-Mendez et al., 2006;
Koohmaraie & Geesink, 2006; Koohmaraie et al., 2002). Lametsch et al.
(2003, 2002) also show several of these proteins (enolase and
creatine kinase) changing in post mortem pork. Indeed, post mortem
degradation of creatine kinase has been observed in several studies
(Lametsch et al., 2002; Purintrapiban et al., 2001; Stoeva, Byrne,
Mullen, Troy, & Voelter, 2000; Troy et al., 1997). We observed that
glyceraldehyde-3-phosphate dehydrogenase significantly increased
across days post mortem (Figs. 2 and 4) as found by other authors
(Laville et al., 2009; Stoeva et al., 2000) who proposed it as an
indicator of the conditioning, i.e. tenderisation, process of ageing
muscle. HSP 70 plays a central role in the cellular response to
apoptotic processes (Liu, Gampert, Nething, & Steinacker, 2006). HSP
70 abundance also increased across the post mortem period. These
may relate to its anti-apoptotic role which is protective against
cellular stress (Arrigo, 2005; Beere, 2004; Beere, 2005). Apoptosis is
thought to start within a few minutes after death and to carry on over
the entire storage period, even at low temperature (Ouali et al., 2006).
If HSPs slow down the process of cellular death, they may slow down
the meat ageing process (Beere, 2004; Ouali et al., 2006).
Only one band (band 5) was significantly associated with waterholding capacity phenotype. Within this band, a number of peptides
which could be assigned to the heat shock protein family were
identified, in particular HSP 70. HSPs are a large family of highly
conserved proteins (Feder & Hofmann, 1999; Gething, 1997). The
HSPs are the most highly induced proteins of the cellular stress
response in mammalian cells (González, Hernando, & Manso, 2000).
They protect cells from stress, restore the function of damaged
proteins and prevent protein aggregation and denaturation (Marruchella et al., 2004; Pelham, 1986). HSP 70 is one of the most
abundant and best-characterized HSPs (Kiang & Tsokos, 1998) and
is known to interact with denatured or misfolded proteins, helping
them in the process of refolding or degrading them (Ciocca,
Oesterreich, Chamness, MCGuire, & Fuqua, 1993; Hartman &
Gething, 1996; Voellmy, 1996). The higher abundance of proteins
in band 5 in DFD and LDrip muscle and the converse lower
abundance in PSE and HDrip muscle could be explained by a
protective effect against protein denaturation and aggregation in the
first two phenotypes, due to the increased abundance of HSP 70
(Bao, Sultan, Nowakc, & Hartung, 2008; Marruchella et al., 2004;
Pelham, 1986). This finding is supported by Yu et al. (2009) who
found a correlation between increased drip loss in longissimus
muscle caused by transport stress and a decline in HSP abundance.
Alternatively, because HSP 70 in stressed tissue is localized in the
nucleus (less easy to extract by centrifugation) while in non
stressed condition it is located in the cytoplasm (easier to extract by
centrifugation), differential localisation of the protein in relation to
cellular stress could explain the higher abundance of this protein in
the drip of high WHC animals. Whatever the mechanism, the results
of this study indicate that a high level of HSP 70 or related proteins
in centrifugal drip may have potential as biomarkers for moderate
to good WHC, though they may not be suitable to discriminate
between DFD and non-DFD meat.
5. Conclusion
Reducing the variability in meat quality is very important for both
consumers and industry (Troy & Kerry, 2010). Great opportunities
exist to improve pork meat quality with the application of tools as
genomics and proteomics. We compared protein abundance of
diverse WHC phenotypes and across time-points post mortem and
9
identified several significant associations between the protein/
fragment band volumes that can be related to WHC or post mortem
protein degradation. The centrifugal drip is reproducible, rich in
proteins and technically straightforward to prepare and this study
reveals it can provide insights into the pathways and processes
underlying meat quality (Hollung et al., 2007). Highlighted proteins
such as HSP 70 could have potential for inclusion in biomarker panels
for the early prediction of meat quality in an industrial setting.
Acknowledgements
This research was funded by the Food Institutional Research
Measure (FIRM) through the Department of Agriculture, Food and
Fisheries in Ireland. Access to and use of instrumentation of the UCD
Conway Mass Spectrometry Resource is gratefully acknowledged. The
authors also wish to acknowledge the Teagasc Walsh Fellowship
scheme and Paula Reid for providing assistance with statistical
analysis.
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Centrifugal drip is an accessible source for protein indicators of pork

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