proteomes
Article
On the Rate of Synthesis of Individual Proteins
within and between Different Striated Muscles of
the Rat
Stuart Hesketh, Kanchana Srisawat, Hazel Sutherland, Jonathan Jarvis and Jatin Burniston *
Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, L3 3AF, UK;
S.J.Hesketh@2014.ljmu.ac.uk (S.H.); K.Srisawat@2015.ljmu.ac.uk (K.S.); H.Sutherland@ljmu.ac.uk (H.S.);
J.C.Jarvis@ljmu.ac.uk (J.J.)
* Correspondence: j.burnistion@ljmu.ac.uk; Tel.: +44-151-904-6265
Academic Editor: Jacek R. Wisniewski
Received: 16 February 2016; Accepted: 10 March 2016; Published: 15 March 2016
Abstract: The turnover of muscle protein is responsive to different (patho)-physiological conditions
but little is known about the rate of synthesis at the level of individual proteins or whether this
varies between different muscles. We investigated the synthesis rate of eight proteins (actin, albumin,
ATP synthase alpha, beta enolase, creatine kinase, myosin essential light chain, myosin regulatory
light chain and tropomyosin) in the extensor digitorum longus, diaphragm, heart and soleus of
male Wistar rats (352 ˘ 30 g body weight). Animals were assigned to four groups (n = 3, in each),
including a control and groups that received deuterium oxide (2 H2 O) for 4 days, 7 days or 14 days.
Deuterium labelling was initiated by an intraperitoneal injection of 10 µL/g body weight of 99.9%
2 H O-saline, and was maintained by administration of 5% (v/v) 2 H O in drinking water provided
2
2
ad libitum. Homogenates of the isolated muscles were analysed by 2-dimensional gel electrophoresis
and matrix-assisted laser desorption ionisation time of flight mass spectrometry. Proteins were
identified against the SwissProt database using peptide mass fingerprinting. For each of the eight
proteins investigated, the molar percent enrichment (MPE) of 2 H and rate constant (k) of protein
synthesis was calculated from the mass isotopomer distribution of peptides based on the amino
acid sequence and predicted number of exchangeable C–H bonds. The average MPE (2.14% ˘ 0.2%)
was as expected and was consistent across muscles harvested at different times (i.e., steady state
enrichment was achieved). The synthesis rate of individual proteins differed markedly within each
muscle and the rank-order of synthesis rates differed among the muscles studied. After 14 days the
fraction of albumin synthesised (23% ˘ 5%) was significantly (p < 0.05) greater than for other muscle
proteins. These data represent the first attempt to study the synthesis rates of individual proteins
across a number of different striated muscles.
Keywords: deuterium oxide; stable isotope labelling; mass isotopomer distribution analysis;
matrix-assisted laser desorption ionisation mass spectrometry; 2D gel electrophoresis; skeletal muscle;
cardiac muscle; protein synthesis
1. Introduction
Skeletal muscle accounts for ~40% of adult body mass and, in addition to its obvious function in
movement, muscle has important roles in glucose homeostasis and protein metabolism. For example, in
healthy individuals muscle is responsible for >75% of insulin-mediated glucose disposal and ~60% of
total body protein turnover. Therefore, maintaining adequate muscle mass and function are important
factors that impact long-term health. Age-related losses in muscle mass lead to frailty and loss of
independence, and so directly impact an individual’s quality of life. Moreover, those individuals
with the greatest level of muscle loss are more likely to be obese or suffer from metabolic disorders
Proteomes 2016, 4, 12; doi:10.3390/proteomes4010012
www.mdpi.com/journal/proteomes
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including impaired glucose tolerance [1]. Furthermore, because skeletal muscle is the major repository of
protein/amino acids that can be mobilised during starvation or injury, muscle loss is also an independent
predictor of mortality in cachexia associated with diseases such as cancer and chronic heart failure [2].
Muscle mass is determined by the historical balance between the synthesis and degradation of its
constituent proteins. Synthesis of new protein in vivo has traditionally been investigated by metabolic
labelling using tracers such as the stable isotope labelled amino acid L-[ring-13 C6 ]-phenylalanine e.g., [3].
This enables the fraction of newly synthesised protein to be calculated from the precursor: product
ratio. Incorporation of stable-isotope labelled amino acids in to protein in vivo is most commonly
combined with gas-chromatography mass spectrometry (GC-MS) analysis of hydrolysed amino acids
to measure the average rate of synthesis in protein mixtures extracted from skeletal muscle of humans
and non-human animal models [4]. In rats, the average half-life of proteins is approximately 14 days in
mixed-fibre locomotive muscles, whereas in slow-twitch postural muscles protein half-life is estimated to
be 7 days [5]. These values represent averages across the entire proteome and so are a gross simplification
of events at an individual protein level. Indeed the average turnover rate may be misleading because
turnover of the mitochondrial protein, cytochrome c, is more rapid in fast- than slow-twitch muscle [6].
Protein synthesis and degradation are each intricate processes that are regulated independently
and on a protein by protein basis to maintain homeostasis or to facilitate muscle adaptation. A greater
understanding of synthesis rates at the individual protein level could significantly advance knowledge
regarding differences in muscle phenotype and the adaptation of muscle to physiological and
pathophysiological stimuli. Watt et al. [7] reports differences between fast-twitch muscle extensor
digitorum longus (EDL) and slow-twitch soleus in rats during a 2-week regimen of high-intensity
jump training. The growth rate of both EDL and soleus increased up to 70% when compared to control,
but growth of the fast-twitch muscle was primarily achieved through greater (28%) protein synthesis,
whereas in the soleus muscle protein synthesis did not change but there was a 38% decrease in protein
degradation. While it is likely some proteins may not have followed these overall trends, until recently
it has not been possible routinely to measure isotope incorporation (i.e., synthesis) in large numbers
of individual proteins. However, the application of proteomic separation techniques in striated
muscle [8] along with advances in the sensitivity of mass spectrometers now enable such work to be
undertaken. For example, Jaleel et al. [9] reports metabolic labelling with L-[ring-13 C6 ]-phenylalanine
in vivo and analysis of muscle mitochondria using 2-dimensional gel electrophoresis (2DGE). Gel spots
were identified by peptide mass spectrometry, whereas incorporation of the stable isotope label was
measured in mixtures of hydrolysed amino acids using GC-MS. The marriage of these established
techniques enabled synthesis rates to be calculated for 68 mitochondrial proteins in rat skeletal muscle.
Jaleel et al. confirms the synthesis of proteins within a muscle differs on a protein by protein basis.
Of the proteins investigated beta enolase had the greatest synthesis rate (11%/day) while myosin light
chain regulatory had the lowest (3%/day), indicating an approximate 10-fold variation in protein
synthesis rates across the proteins investigated.
A major shortcoming of amino acid tracer studies is that it is impractical to measure the true
enrichment of the precursor pool (i.e., aminoacyl-tRNA) directly. This combined with uncertainties
regarding the transport of amino acids between intracellular and extracellular compartments, and
recycling of amino acids from protein degradation, gives rise to uncertainty regarding the calculation
of the renewal rate of the protein products. Secondly, amino acid tracers require intravenous
infusion, which is invasive and therefore necessarily restricts the duration of such investigations.
As a consequence, it is likely the average synthesis rates calculated from mixtures of muscle proteins
are skewed toward that of the more rapidly synthesised proteins. Moreover, linear extrapolation
of short-term (e.g., 30 min) incorporation of an amino acid tracer e.g., [10] to longer time periods
(e.g.,%/day) will further lead to overestimation because incorporation of labelled amino acid in to
the protein product exhibits first-order (rise-to-plateau) kinetics. These shortcomings can be entirely
overcome through the use of deuterium oxide (2 H2 O or “D2 O”) to label macromolecular precursors,
including amino acids, in vivo [11].
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2H O
2
has the fundamental advantage that it can be administered via drinking water and labelling
can be conducted in free-living animals over experimental periods spanning days or months [12].
2 H O equilibrates within the body water compartment rapidly (<30 min; in rodents) and H–C bonds
2
of amino acids (principally alanine) become 2 H-labelled intracellularly via transamination and/or de
novo synthesis [13]. Therefore, unlike amino acid tracers, enrichment of the precursor pool is not
affected by amino acid metabolism, membrane transport or dilution effects from protein degradation.
After incorporation in to newly synthesised protein the 2 H-label is irreversible [14] and can be quantified
by GC-MS of amino acid hydrolysates (e.g., [14]) or by peptide mass spectrometry of tryptic digests.
For example, Xiao et al. [15] reports a method for determining protein synthesis using 2 H2 O labelling
in vivo and matrix-assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF
MS) of rat serum proteins. We have used a similar approach to investigate the synthesis of eight proteins
in four striated muscles, including the heart, diaphragm, extensor digitorum longus (EDL) and soleus.
Striated muscles are highly specialised tissues that exhibit a broad range of different phenotypic
properties particularly with regard to speed of contraction and fatigue resistance [16]. The diversity
of muscle phenotypes is largely due to differential expression of different isoforms of key contractile
proteins, which concomitantly dictates the relative abundance of enzymes involved in substrate
utilisation and energy production. In the past, we have reported the relative abundance of proteins
in various rat striated muscles using proteomic techniques including 2DGE and MALDI-TOF MS
e.g., [17–19] but there is currently a lack of equivalent data regarding synthesis rates of rat muscle
proteins. In particular, we sought to investigate whether the rank order of synthesis rates for a selection
of proteins is similar across different striated muscles or if enzymes that exist at a greater concentration
in a particular muscle also exhibit a more rapid rate of renewal.
2. Materials and Methods
Experimental procedures were conducted under the British Home Office Animals (Scientific
Procedures) Act 1986 and were approved by the local ethical review committee. Male Wistar rats
(352 ˘ 30 g body weight) were bred in-house in a conventional colony, housed in controlled conditions
of 20 ˝ C, 45% relative humidity, and a 12 h light (0600–1800 hours) and 12 h dark cycle, with water and
food available ad libitum.
Animals were assigned to four groups (n = 3, in each), including a control group and three
groups that received deuterium oxide (2 H2 O) for either 4 days, 7 days or 14 days. Deuterium
administration was initiated by an intraperitoneal injection of 10 µL.g 99% 2 H2 O-saline, and was
maintained by administration of 5% (v/v) 2 H2 O in drinking water, which was refreshed daily. Animals
were asphyxiated with a rising concentration of CO2 and killed by cervical dislocation. Samples of the
heart (HRT) and diaphragm (DIA), and the entire extensor digitorium longus (EDL) and soleus (SOL)
were isolated. Each muscle was cleaned of fat and connective tissue and then weighed before being
frozen in liquid nitrogen and stored at ´80 ˝ C.
Muscles were ground under liquid nitrogen and a portion (~100 mg) homogenised on ice in
10 volumes of 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris pH 7.4 including phosphatase
inhibitor and complete protease inhibitor cocktails (Roche, Indianapolis, USA). After centrifugation at
12,000ˆ g, 4 ˝ C for 45 min the supernatant was decanted and the protein concentration of a 5 µL aliquot
measured using a modified “microtitre plate” version of the Bradford assay (Sigma, Poole, Dorset, UK).
Muscle homogenates were prepared for 2-dimensional gel electrophoresis (2DGE) as described
previously [18]. An aliquot of each supernatant was precipitated in acetone and resuspended in
7 M urea, 2 M Thiourea, 2% (w/v) CHAPS, 20 mM dithiothreitol, 0.5% (v/v) ampholytes. Samples,
containing 250 mg protein, were loaded on to 13 cm pH 3–11 nonlinear IPG strips (GE Healthcare,
Chalfont St Giles, UK) and focused using an “active rehydration” and isoelectric focusing protocol
comprising: 150 Vh at 30 V, 300 Vh at 60 V, 500 Vh at 500 V, 1000 Vh at 1000 V and 48 000 Vh at 8000 V;
conducted on an IPGPhor II (GE Healthcare) at 20 ˝ C, maximum 50 mA per strip. IPG strips were
equilibrated in 50 mM Tris-HCl pH 8.8, containing 6 M urea, 30% (v/v) glycerol, 70 mM SDS and a
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trace of bromophenol blue. DTT (65 mM) was present as a reducing agent in the first equilibration
and iodoacetamide (135 mM) in the second. Proteins were electrophoresed through 16 cm linear 12%
polyacrylamide gels at 20 ˝ C; at a constant current of 15 mA per gel for 30 min, then 30 mA per gel
until the tracking dye reached the bottom edge of the gel. Gels were washed and stained with colloidal
Coomassie blue (Bio-Safe; BioRad, Hercules, CA, USA) according to the manufacturer’s instructions.
Gel spots were cut and processed using an Xcise robot (Proteome Systems, North Ryde, Australia)
directed by the gel analysis software. Gel plugs destained in three changes of 25 mM ammonium
bicarbonate in 50% acetonitrile were dehydrated before being incubated with 35 µL of 1.25 mg/mL
porcine trypsin (Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate. Peptide solutions
were de-salted and concentrated (Zip-tips; Millipore, Billercia, MA, USA) before being mixed with
matrix (5 mg/mL α-cyano-4-hydroxcinnamic acid in 50:50 acetonitrile and 0.1% trifluoroacetic acid)
and spotted on to 384-well stainless steel target plates. A calibration mix (Laserbio Labs, Sophia
Antipolis, France) consisting of angiotensin II (m/z 1046.2), angiotensin I (m/z 1296.5), neurotensin
(m/z 1672.9), ACTH fragment {1–17} (m/z 2093.5) and ACTH fragment {18–39} (m/z 2465.19) was
mixed 1:1 with matrix solution and spotted (0.5 µL) between every four sample-wells. Peptide
mass spectra were recorded using a matrix-assisted laser desorption ionisation tandem time of
flight (MALDI-TOF/TOF) mass spectrometer (Axima TOF2 ; Shimadzu Biotech, Manchester, UK)
in positive reflectron mode over a mass/charge (m/z) range of 900–3000. Data were smoothed
(Gaussian, 2 chan peak width), baseline subtracted (100 chan peak width) and an adaptive (8.0ˆ)
threshold applied. Peptide mass lists (restricted to 20 peptides over 900–3000 m/z) were produced using
the peak selection tool of the instrument’s Launchpad software (Version 2.8.4) and searched against
the Swiss-Prot database restricted to “Rattus” using the online MASCOT (www.matrixscience.com)
server. The enzyme specificity was set as trypsin allowing one missed cleavage, carbamidomethyl
modification of cysteine (fixed), oxidation of methionine (variable) and an m/z error of ˘0.3 Da.
The amount of newly synthesised protein was calculated in deuterium labelled samples using mass
isotopomer distribution analysis [20]. Peptides were resolved to mass isotopomer envelopes. The pattern
of mass isotopomers from deuterium-enriched samples was used to calculate the molar percent
enrichment (MPE) of deuterium (2 H) in the precursor pool (p) as well as the fraction of newly synthesised
protein. Essentially, the enrichment of “heavy” isotopomers (e.g., m2 /m1 ) provides information on
the level of 2 H in the precursor pool, whereas the incorporation of 2 H “heavy” isotopes in to newly
synthesised protein causes the relative abundance of the monoisotopic peak (m0 ) to decline. Raw mass
spectra were exported in mzXML format and mMass software (Version 5.5.0, http://www.mmass.org)
was used to measure the intensities of mass isotopomers (m0 , m1 , m2 ) of peptides of interest, and to
calculate the elemental composition of each peptide. The number (N) of exchangeable H–C bonds in each
peptide was estimated from [21] and subsequent data handling was performed in R (www.R-project.org).
Mass isotopomer intensities were converted to relative abundance of each mass isotopomer envelope
and mass isotopomer distribution analysis (MIDA) tables were created using multinomial distribution,
reported in detail in [20]. When the number of exchangeable H–C bonds and the elemental composition
of the peptide are known, the level of precursor enrichment can be calculated from the ratio of enrichment
between “heavy” isotopomers (i.e., em2 /em1 ). When the level of precursor enrichment is known the
rise in the m1 /m0 ratio due to 2 H incorporation can be used to calculate fractional synthesis. Fractional
synthesis was calculated from the slope of the m1 /m0 regression line created from modelled data of each
peptide based on its natural distribution of C, H, N, O elements, the number of exchangeable H–C bonds
and the measured enrichment of 2 H in the precursor pool.
Statistical analyses were conducted in Prism v6.02/6.0c (GraphPad, La Jolla, CA, USA); nonlinear
one-phase association was used to estimate the rate constant k and half-life of each protein based on
the amount of newly synthesised protein measured after 4 days, 7 days and 14 days of deuterium
administration. The coefficient of determination (R2 value) was used as a measure of the goodness of
the fitted non-linear regression and one-way ANOVA was used to investigate significant differences of
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individual protein synthesis rates across muscle tissues. The level of statistical significance was set at
p < 0.05.
3. Results
Protein synthesis in vivo was investigated in the diaphragm, heart, EDL and soleus of rats
administered 2 H2 O for either 4 days, 7 days or 14 days. Control (0-day) muscles were harvested
from animals that did not receive deuterium. There was no significant difference in the wet weight of
muscles harvested at each of the time points investigated.
Muscle homogenates were separated by 2DGE (Figure 1) and eight spots matched across each
muscle were processed for mass spectrometry. Peptide mass fingerprinting (Table 1) was used to
confirm the identification of albumin (ALBU), actin (ACTS), β-enolase (ENOB), muscle creatine kinase
(KCRM), ATP synthase-α (ATPA), tropomyosin α-1 (TPM1), essential myosin light chain (MYL3) and
regulatory myosin light chain (MLRV). Soleus muscle samples were used to investigate synthesis of
different species of ALBU, ENOB and KCRM that each exhibited similar relative molecular mass but
different isoelectric points.
Peptides selected for mass isotopomer distribution analysisβ (MIDA) were of relatively high intensity
and signal:noise ratio, rich in alanineαand were specific to
α the identified protein. With the exception
of ATPA (three peptides), five peptides were analysed for each protein. Table 1 provides details of
the sequence, monoisotopic peak and elemental composition of the selected peptides. The number
of exchangeable H–C bonds was estimated from published data on tritium incorporation in to amino
acids in vivo [21]. Figure 2 illustrates changes to the pattern of peptide mass isotopomers due to the
incorporation of 2 H during protein synthesis in vivo. Peptides were resolved to mass isotopomer
envelopes consisting of the monoisotopic (m0 ) peak, and “heavy” isotopomer peaks (m1 , m2 , m3 ).
The ratio between “heavy” isotopomers (e.g., m2 /m1 ) provides information on the enrichment of 2 H in
the precursor pool. The incorporation of 2 H “heavy” isotopes in to newly synthesised protein causes the
relative abundance of the monoisotopic peak (m0 ) to decline and the ratio of m1 /m0 to increase. When
the level of precursor enrichment is known the rise in the m1 /m0 ratio due to 2 H incorporation can be
used to calculate synthesis.
incorporation can be used to calculate synthesis.
Figure 1. Separation of muscle proteins by 2-dimensional gel electrophoresis.
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Whole muscle homogenates resolved by 2-dimensional gel electrophoresis and stained with
colloidal Coomassie blue. Protein identities were confirmed by peptide mass fingerprinting (Table 1)
of in-gel digests analysed by matrix-assisted laser desorption ionisation mass spectrometry.
Table 1. Protein identification by peptide mass fingerprinting.
Protein
ALBU
ACTS
KCRM
Score
170
86
117
Coverage
Peptide Sequence
m/z
CHNO
H–D
27%
CPYEEHIK
FKDLGEQHFK
FPNAEFAEITK
LGEYGFQNAVLVR
DVFLGTFLYEYSR
1075
1248
1266
1465
1609
C45H67N11O14
C58H85N15O16
C59H87N13O18
C67H104N18O19
C77H108N16O22
17
13
21
18
17
28%
AGFAGDDAPR
GYSFVTTAER
AVFPSIVGRPR
QEYDEAGPSIVHR
SYELPDGQVITIGNER
976
1130
1198
1500
1791
C41H61N13O15
C50H75N13O17
C55H91N17O13
C64H97N19O23
C77H123N21O28
25
17
23
29
26
30%
FEEILTR
GYTLPPHCSR
DLFDPIIQDR
SFLVWVNEEDHLR
GTGGVDTAAVGAVFDISNADR
907
1187
1231
1643
1992
C42H66N10O13
C49H75N15O14
C55H86N14O18
C75H110N20O22
C83H113N25O32
14
19
15
21
39
1166
1556
1572
1804
1901
C54H83N15O14
C70H109N17O21
C70H109N17O21
C79H129N21O27
C86H137N19O23
19
26
26
42
26
ENOB
124
39%
IGAEVYHHLK
VVIGMDVAASEFYR
VVIGMDVAASEFYR
AAVPSGASTGIYEALELR
LAMQEFMILPVGASSFK
ATPA
137
34%
AVDSLVPIGR
GIRPAINVGLSVSR
TGAIVDVPVGDELLGR
1026
1438
1611
C45H79N13O14
C62H111N21O18
C70H119N19O24
19
31
28
38%
HIAEDADR
LDKENALDR
LVIIESDLER
KATDAEADVASLNR
SIDDLEDELYAQK
926
1073
1186
1460
1538
C37H59N13O15
C44H76N14O17
C52H91N13O18
C59H101N19O24
C59H101N19O24
23
17
19
31
23
44%
HVLATLGER
EAFLLFDR
DQGGYEDFVEGLR
DTGTYEDFVEGLR
NKDTGTYEDFVEGLR
995
1010
1484
1501
1744
C43H74N14O13
C48H71N11O13
C64H93N17O24
C65H96N16O25
C75H114N20O28
18
15
23
21
22
63%
VFDPEGKGSLK
DGFIDKNDKR
EAFTIMDQNR
NLVHIITHGEEKD
LKGADPEETILNAFK
1176
1192
1240
1504
1645
C53H85N13O17
C51H81N15O18
C51H81N15O18
C65H105N19O22
C74H120N18O24
17
13
15
21
26
TPM1
MYL3
MLRV
163
91
119
Albumin (ALBU), skeletal muscle alpha actin (ACTS), skeletal muscle creatine kinase (KCRM), beta-enolase
(ENOB), ATP synthase alpha (ATPA), tropomyosin alpha-1 (TPM1), myosin essential light chain 3 (MYL3),
myosin regulatory light chain slow/ventricular (MLRV). Score (MOWSE) and coverage (percent sequence
coverage) reported from peptide mass fingerprinting and mascot searches against the Rattus Swiss-Prot database.
Monoisotopic peak (m/z), elemental composition (CHNO) and number of exchangeable H–C bonds (H–D) are
reported for each peptide.
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Figure 2. Mass spectrometry of deuterium-labelled peptides. Peptides were resolved as series of mass
isotopomers (m0 , m1 , m2 , ...) using matrix-assisted laser desorption ionisation mass spectrometry
(MALDI-MS). Experimental mass spectra (red traces) from peptide DGFIDKNDLR (residues 41–50 of
slow/ventricular myosin regulatory light chain; MLRV) are displayed from samples taken after 0, 4, 7 or
14 days of deuterium oxide (2 H2 O) administration in vivo. The blue trace represents the distribution of
mass isotopomers predicted from the elemental composition of the peptide and the natural abundances
of 12 C, 1 H, 14 N and 16 O (mMass software). The m0 (monoisotopic) peak is composed entirely of
primary isotopes (i.e., 12 C, 1 H,14 N and 16 O), whereas m1 , m2 , m3 contain either 1, 2 or 3 “heavy”
isotopes (e.g., 13 C, 2 H, etc.).
Fractional synthesis was calculated from the slope of the m1 /m0 regression line created from
modelled data, which is equivalent to the use of MIDA tables reported previously [20]. Briefly,
regression was based on the natural distribution of C, H, N, O elements, the number (N) of exchangeable
H–C bonds (H–D in Table 1) and the molar percent enrichment (MPE) of 2 H in the precursor pool (p).
Table 2 provides an example of data modelled from peptide DGFIDKNDLR displayed in Figure 2.
To illustrate the method in this example the molar fraction of mass isotopomers was calculated
using multinomial distribution based on the natural abundance of 13 C only and the rate of protein
degradation was assumed to be constant. Two different scenarios have been modelled. In Table 2a,
differences in the fraction (%) of newly synthesised protein can be seen to alter the ratio between
“heavy” and “light” (e.g., m1 /m0 ) isotopomers while the pattern (ratio) of enrichment between “heavy”
isotopomers (i.e., em2 /em1 ) remains constant. Conversely, in Table 2b, enrichment of 2 H in the
precursor pool is varied between 0 and 2.5% while synthesis is held constant (100%). Under these
conditions the pattern of enrichment of “heavy” isotopomers (i.e., em2 /em1 ) changes in accordance
with the level of precursor enrichment. This ability to distinguish between the effects of protein
synthesis and precursor enrichment is fundamental to the use of MIDA.
Table 2. (a) Mass isotopomer distribution analysis (MIDA) model of the effects of protein synthesis at a
fixed precursor enrichment of 2.5%. (b) MIDA model of the effects of precursor enrichment a fixed
−
−
level (100%) of synthesis.
(a)
−
−
−
−
−
−
Synthesis (%)
m0
m1
m2
m1/m0
m2/m1
em0
em1
em2
em1/em0
em2/em1
0
25
50
75
100
0.5797
0.5440
0.5082
0.4725
0.4368
0.3288
0.3450
0.3611
0.3772
0.3933
0.0914
0.1110
0.1307
0.1503
0.1699
0.5672
0.6341
0.7105
0.7983
0.9006
0.2781
0.3219
0.3619
0.3984
0.4319
–
´0.0357
´0.0715
−´0.1072
−´0.1430
–
0.0161
0.0323
0.0484
0.0645
–
0.0196
0.0392
0.0588−
0.0784−
–
´0.4512
´0.4512
´0.4512
´0.4512
–
1.2162
1.2162
1.2162
1.2162
−
−
−
−
−
−
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Table 2. Cont.
(b)
Precursor (%)
m0
m1
m2
m1/m0
m2/m1
em0
em1
em2
em1/em0
em2/em1
0
0.5
1.0
1.5
2.0
2.5
0.5797
0.5472
0.5168
0.4883
0.4617
0.4368
0.3288
0.3461
0.3610
0.3737
0.3844
0.3933
0.0914
0.1067
0.1223
0.1380
0.1539
0.1699
0.5672
0.6326
0.6986
0.7652
0.8325
0.9006
0.2781
0.3082
0.3387
0.3694
0.4005
0.4319
–
´0.0325
´0.0630
´0.0914
´0.1180
´0.1430
–
0.0173
0.0321
0.0448
0.0555
0.0645
–
0.0152
0.0308
0.0466
0.0625
0.0784
–
´0.5315
´0.5105
´0.4902
´0.4705
´0.4512
–
0.8816
0.9587
1.0399
1.1256
1.2162
Predicted molar fraction of mass isotopomers (m0, m1, m2) at 0% synthesis (2a) or 0% precursor enrichment
(2b) is subtracted from the molar fraction of mass isotopomers that include deuterium to give the enriched
molar fraction of mass isotopomers (em0, em1, em2). In Table 2a, m1/m0 ratio is linearly associated with the
fraction of newly synthesised protein. In Table 2b, the em2/em1 ratio is linearly associated with the level of
precursor enrichment; the slope of these relationships is determined by the number of exchangeable C–H sites
and the intercept is principally determined by the number of C atoms in the peptide. For example, the straight
line equation of em2/em1 for peptide DGFIDKNDLR (C–H = 13, C = 51) is y = 0.1672x + 0.7936.
Figure 3. Time course of newly synthesised protein in vivo. The incorporation of deuterium in vivo was
used to measure the synthesis of new protein using mass isotopomer distribution analysis. Data are
presented as the average (Mean ˘ SEM) percentage of newly synthesised protein measured from
3–5 peptides for each protein, replicated in n = 3 biological samples. Data are fitted using a non-linear
first-order equation. The range of the y-axis is consistent across muscles for each protein (i.e., by row)
by differs between proteins (i.e., by column).
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MPE of the precursor pool was calculated from the enrichment of the m2 /m1 isotopomers of a
selection of the best quality peptides (n = ~80). The average MPE from all peptides of 2 H2 O treated
animals was 2.14% ˘ 0.2% and did not significantly differ between animals or across the time points
(4 days, 7 days, 14 days) investigated. This is consistent with previous investigations, demonstrating
rapid (<30 min in rats) equilibration of deuterium oxide in the body water compartment [13].
Mass spectra were collected from each of the selected gel spots (Figure 1) in each striated muscle
harvested from control animals and rats that received 2 H2 O for either 4 days, 7 days or 14 days (n = 3
in each group). The fraction of newly synthesised protein after 4, 7 and 14 days calculated by MIDA
was fitted using nonlinear first-order regression (Figure 3) to estimate the rate constant (k) of protein
synthesis and the half-time of protein renewal (Table 3) assuming no change in protein abundance.
Table 3. Half-time and rate constants (k) of protein synthesis in rat striated muscles calculated from
14 days 2 H2 O administration in vivo.
Protein
Heart
Diaphragm
EDL
Soleus
Albumin (ALBU)
Actin (ACTS)
Creatine kinase (KCRM)
Beta enolase (ENOB)
ATP synthase α (ATPA)
Tropomyosin (TPM1)
Myosin essential light chain (MLY3)
Myosin regulatory light chain (MLRV)
11.0 (k = 0.063)
20.2 (k = 0.034)
NA
NA
NA
28.5 (k = 0.024)
10.8 (k = 0.064)
25.3 (k = 0.027)
11.0 (k = 0.063)
28.7 (k = 0.024)
7.8 (k = 0.089)
21.1 (k = 0.033)
25.2 (k = 0.028)
NA
6.5 (k = 0.107)
NA
14.4 (k = 0.048)
87.8 (k = 0.008)
8.4 (k = 0.083)
24.2 (k = 0.029)
28.9 (k = 0.024)
7.4 (k = 0.094)
9.3 (k = 0.074)
47.8 (k = 0.016)
13.3 (k = 0.052)
NA
NA
-
Half-time in days and rate constant (k) of protein synthesis calculated from nonlinear regression of the change in
newly synthesised protein measured at 4 days, 7 days and 14 days using mass isotopomer distribution analysis
of deuterium-labelled muscles. NA = data not available due to inadequate data fitting.
The percentage of newly synthesised protein in rat striated muscles after 14 days of 2 H2 O
administration (Table 4) spanned from 0.5% ˘ 0.6% (heart ATP synthase alpha) to 29.2% ˘ 8.4%
(diaphragm albumin). In each muscle investigated the synthesis of albumin was significantly (p < 0.05)
greater than that of the other muscle proteins analysed. With the exception of albumin, the rank order
of synthesis (Figure 4) was different across each of the muscles investigated. Moreover, in the soleus
muscle, the synthesis of ALBU spot (i) was significantly (p < 0.05) greater than ALBU spot (ii), which
provides evidence of a functional difference between these protein species.
Table 4. Percentage of newly synthesised protein in rat striated muscles after 14 days 2 H2 O administration
in vivo.
Protein
Heart
Diaphragm
EDL
Soleus
Albumin (ALBU)
Actin (ACTS)
Creatine kinase (KCRM)
Beta enolase (ENOB)
ATP synthase α (ATPA)
Tropomyosin (TPM1)
Myosin essential light chain (MLY3)
Myosin regulatory light chain (MLRV)
17.2 ˘ 3.0 *
2.0 ˘ 0.9
2.5 ˘ 1.2
5.5 ˘ 2.8
0.5 ˘ 0.6
2.7 ˘ 1.2
5.8 ˘ 1.5
13.1 ˘ 5.3
29.2 ˘ 8.4 *
4.1 ˘ 1.3
8.4 ˘ 2.3
5.5 ˘ 1.4
5.6 ˘ 2.3
3.0 ˘ 2.6
2.2 ˘ 3.0
12.2 ˘ 1.7
25.1 ˘ 2.5 *
1.5 ˘ 0.6
3.0 ˘ 1.5
6.9 ˘ 3.2
13.2 ˘ 7.2
2.7 ˘ 2.1
11.4 ˘ 3.6
10.7 ˘ 2.3
20.6 ˘ 8.4 *
9.5 ˘ 4.2
15.2 ˘ 5.1
-
Percentage of newly synthesised protein measured after 14 days deuterium oxide labelling in vivo. * p < 0.05,
significantly different from other Proteins. All data are presented as Mean ˘ SEM of n = 3–5 peptides from each
protein measured in n = 3 animals.
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Figure 4. Rank order of newly synthesised protein within each striated muscle. Data represent
percentage (mean ˘ SD) of new protein synthesised after 14 days, presented in relative rank order
within the heart, diaphragm, extensor digitorium longus and soleus.
4. Discussion
We report novel data regarding the synthesis of eight proteins in four striated muscles of the rat
using deuterium oxide labelling in vivo, peptide mass spectrometry and mass isotopomer distribution
analysis (MIDA; [20]). To the best of our knowledge, this is the first work to report the synthesis of
individual proteins across a range of different rat striated muscles. One of our major findings was that
the amount of protein synthesis measured here in free-living animals across a number of time points
during a 14-day period was generally less than reported in previous studies that used short-term
(e.g., 30 min) infusion of an amino acid tracer.
In the current work the synthesis of albumin (~23% after 14 days) in each of the muscles
investigated was significantly greater than the range (0.5%–15.2% after 14 days) exhibited by the
other muscle proteins. Albumin is a prominent feature of the skeletal muscle proteome [22] but
expression of the albumin gene is low in muscle [23]. Therefore, our findings support the conclusion
that albumin extracted from muscle is primarily synthesised in the liver, which typically has a more
rapid turnover of proteins than striated muscle [24]. After removal of the myofibrillar components,
albumin is one of the most abundant proteins in skeletal muscle [22,25]. Therefore, our data have
consequences for the interpretation of previously reported synthesis measurements of mixed proteins
from myofibrillar, sarcoplasmic and mitochondrial fractions e.g., [26]. The high abundance of albumin
in the sarcoplasmic fraction of muscle is likely to have artificially raised the average synthesis rate
reported for this fraction and may also have masked biologically important changes in the synthesis of
less abundant muscle proteins in response to experimental interventions.
Skeletal muscle albumin is primarily localised to the interstitium [27] and is thought to aid
transport of fatty acids in to muscle. The total abundance of albumin in fast-twitch skeletal muscle
increases during transformation induced by chronic low frequency stimulation [28]. Similarly,
intensity-controlled endurance training also increases the abundance of a specific species of albumin
in the fast-twitch plantaris muscle of rats [18]. In the current work we investigated the synthesis of
different species of three proteins in the soleus muscle (Figure 1), including albumin. The predominant
albumin species {ALBU (i)} had a relatively high level of synthesis (20.6% ˘ 8.4% after 14 days),
consistent with our findings in the other striated muscles (Table 4). In contrast, synthesis of the
more acidic albumin species {ALBU (ii)} was significantly (p < 0.05) less (9.6% ˘ 1.8% after 14 days),
and it is this species {ALBU (ii)} that is specifically increased in rat plantaris muscle in response to
endurance exercise training [18]. The difference in isoelectric point between the ABLU (i) and ALBU
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(ii) species is likely to be due to a post-translational modification, and we show this modification is
able to affect the turnover of the albumin protein. The modification to albumin may occur directly
within the liver or secondarily in the periphery where the shift toward a more acidic isoelectric point
might be associated with lesser uptake/residency in the muscle interstitium. Alternatively, because
albumin mRNA can be detected (albeit at low levels) in skeletal muscle [23] the relatively acidic species
of albumin {ALBU (ii)} might represent protein that was synthesised and modified in the muscle
rather than the liver. In the future, inclusion of hepatic tissue alongside the collection of various
striated muscles from endurance-trained or chronically stimulated animals might resolve some of
these unanswered questions.
Contrary to our findings for species of albumin in the soleus, there was no difference in synthesis
between the two species of either beta-enolase or creatine kinase (Figure 4). This indicates the
post-translational modifications underlying the charge differences in these proteins did not alter
their rate of synthesis. Nonetheless, further investigation encompassing all species from a greater
number of proteins is needed to determine whether this is generally true for the majority of muscle
proteins. Although 2DGE is relatively laborious and has limited scalability, we believe such data
will be important to the interpretation of findings from high-throughput tandem mass spectrometry
(e.g., LC-MS/MS) analyses of protein digests, which afford greater proteome coverage [25] but cannot
distinguish between the different species of each protein. Wider analysis is also required to build a
more complete picture of differences in the synthesis of proteins across different muscles. Our initial
finding is that the rank order of renewal of proteins is different in the myocardium, mixed-fibre
diaphragm, slow-twitch soleus and fast-twitch EDL (Figure 4). Of the two proteins (beta-enolase and
creatine kinase) investigated in the soleus and EDL, the amount of protein synthesised after 14 d
was greater (NS) in the soleus (Table 4), which supports previous data [5,29] from protein mixtures,
supporting the hypothesis that slow-twitch postural muscles have a higher rate of turnover than less
frequently activated fast twitch muscles such as EDL. However, on the whole, we found no simple
relationship between myofibre phenotype/muscle activity pattern and the synthesis of individual
proteins. Of the proteins investigated, the synthesis of tropomyosin (~3% after 14 days) was the most
consistent across the different striated muscles whereas ATP synthase-α exhibited more than a 20-fold
difference between the heart (0.5% after 14 days) and EDL (13% after 14 days). This finding is in
accordance with earlier work [6] reporting that synthesis of the mitochondrial protein, cytochrome c,
is greater in fast- than slow-twitch muscle.
As anticipated, the amount of protein synthesised during a 14-day period was less than predicted
from extrapolation of short-term synthesis measurements. For example, Jaleel et al. [9] reports the
synthesis of TPM1 is 8%/day based on stable isotope incorporation after short-term (~20 min) infusion
of ring-[13 C6 ] phenylalanine in vivo. In the current work, the rate constant for TPM1 was 2.7%/day
(Table 3). Because incorporation of label from the precursor pool in to a newly synthesised protein
exhibits non-linear first-order kinetics, this discrepancy is most likely due to linear extrapolation of
data recorded after 20 min of infusion in [9]. It is also important to recognise that spots excised from
2DGE often contain multiple proteins [30]. Therefore, GC-MS analysis of amino acids hydrolysed from
2DGE spots (e.g., Jaleel et al. [9]) could include contamination from proteins that share the same relative
mass and isoelectric point as the protein of interest. Arguably, the level of contamination may be small
but is nonetheless an unknown variable that is able influence the results. In comparison, our current
approach offers greater selectivity because both precursor enrichment and protein synthesis were
quantified from protein-specific peptides, whereas unidentified peptides (i.e., potential contaminants)
were ignored.
Currently few data exist using comparable proteomic analysis of deuterium-labelled muscle
proteins. Kasumov et al. [31] reports synthesis rates for 28 proteins in intermyofibrillar and
subsarcolemmal mitochondria of the rat heart using 2 H2 O labeling and high-resolution peptide
mass spectrometry. Protein synthesis was greater in the subsarcolemmal mitochondrial population,
therefore, sub-cellular localisation is a further parameter that needs to be considered when investigating
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the synthesis of individual proteins. Kasumov et al. [31] reports the half-life of cardiac ATPA is
approximately 27–30 days which is equivalent to the values estimated here in the diaphragm and EDL
(Table 2). More recently, 2 H2 O labelling in vivo has been used to investigate synthesis in the triceps
of ovariectomised rats exposed to a selective androgen receptor modulator or vehicle control [32].
Rats were administered deuterium oxide for a period of 7 days prior to muscle harvesting and peptide
mass spectrometry was used to calculate the synthesis of individual proteins. Table 5 provides a
comparison between our 7-day data and equivalent data reported in Table 2 of Shankaran et al. [32].
In most cases, we report less protein synthesis in the hindlimb muscles than Shankaran et al. report in
the triceps. The exception is regulatory myosin light chain, which was synthesised similarly in each of
the striated muscles investigated.
Table 5. Percentage of newly synthesised protein in rat striated muscles after seven days D2O
administration in vivo.
Protein
Heart
Diaphragm
EDL
Soleus
Triceps †
Albumin (ALBU)
Actin (ACTS)
Creatine kinase (KCRM)
Beta enolase (ENOB)
ATP synthase α (ATPA)
Tropomyosin (TPM1)
Myosin essential light chain (MLY3)
Myosin regulatory light chain (MLRV)
11.9 ˘ 2.4
1.3 ˘ 0.6
0.4 ˘ 0.3
5.3 ˘ 0.3
0.3 ˘ 0.2
1.6 ˘ 0.7
1.2 ˘ 1.1
7.9 ˘ 3.0
20.2 ˘ 6.1
2.6 ˘ 0.8
5.9 ˘ 1.7
4.0 ˘ 1.0
3.3 ˘ 1.0
1.7 ˘ 1.6
2.2 ˘ 2.1
6.3 ˘ 1.3
16.6 ˘ 2.7
0.8 ˘ 0.5
2.2 ˘ 1.0
4.2 ˘ 1.2
8.1 ˘ 4.8
2.0 ˘ 1.4
7.2 ˘ 2.1
5.6 ˘ 1.1
13.6 ˘ 0.8
1.0 ˘ 0.3
7.5 ˘ 2.6
-
7.0 ˘ 0.3
13.0 ˘ 0.4
13.7 ˘ 0.3
30.0 ˘ 1.2
14.5 ˘ 0.4
13.4 ˘ 1.6
9.6 ˘ 0.3
†
From Table 2 of Shankaran et al. 2015 [32].
An advantage of deuterium oxide labelling compared to infusion of amino acid labelled tracers is
that experiments can be conducted over longer durations and therefore provide a better estimation
of the rate of protein synthesis. We used a minimum number of time points over the duration of
the experiment to fit nonlinear regression curves to our data. However, incorporation of deuterium
(i.e., protein synthesis) did not reach a plateau for any of the proteins investigated during the 14-d
period. Therefore, we report estimated rate constants of protein synthesis (Table 3) of each investigated
protein and stress that it may be inappropriate to extrapolate values to estimate half-time for protein
renewal or time for complete turnover of the protein pool. Instead, in Table 4, we report the fraction
of newly synthesised protein after 14 days to enable a more robust comparison of the synthesis of
individual proteins across the different striated muscles. Based on our current findings, the duration
of future experiments should extended >3 weeks in order to capture a more accurate reflection of the
individual protein synthesis rates within rat striated muscles.
5. Conclusions
Our current work demonstrates that the synthesis of individual proteins differs not only within
a muscle, but also the rank order of which protein exhibits the greatest synthesis is different, depending
on the muscle investigated. We believe this work raises many exciting opportunities for further
investigation. Not least, it will be interesting to discover how different muscle sub-fractions respond
to muscle adaptation and if the rank order of synthesis in muscle is altered in response to important
physiological and patho-physiological perturbations.
Acknowledgments: K.S. is a recipient of a Royal Thai Government Fellowship (CS_4428).
Author Contributions: J.B. conceived and designed the experiments; S.H., H.S., K.S. and J.J. performed the
experiments; S.H., K.S., J.B. and J.J. analyzed the data; S.H., J.J. and J.B. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Abbreviations
The following abbreviations are used in this manuscript:
MPE
GC-MS
EDL
HRT
DIA
SOL
2DGE
2 H O or D O
2
2
MALDI-TOF
MS
ALBU
ACTS
ENOB
KCRM
ATPA
TPM1
MYL3
MLRV
MIDA
m0
LC-MS
Molar percent enrichment
Gas chromatography-mass spectrometry
Extensor digitorium longus
Heart
Diaphragm
Soleus
2 dimension gel electrophoresis
Deuterium oxide
Matrix assisted laser desorption ionization-time of flight
Mass spectrometry
Albumin
Actin
β-enolase
Muscle creatine kinase
ATP synthase-α
Tropomyosin α-1
Essential myosin light chain
Regulatory myosin light chain
Mass isotopomer distribution analysis
Monoisotopic peak
Liquid chromatography-mass spectrometry
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Parr, E.B.; Coffey, V.G.; Hawley, J.A. ‘Sarcobesity’: A metabolic conundrum. Maturitas 2013, 74, 109–113.
[CrossRef] [PubMed]
Springer, J.; Tschirner, A.; Haghikia, A.; von Haehling, S.; Lal, H.; Grzesiak, A.; Kaschina, E.; Palus, S.;
Potsch, M.; von Websky, K.; et al. Prevention of liver cancer cachexia-induced cardiac wasting and heart
failure. Eur. Heart J. 2014, 35, 932–941. [CrossRef] [PubMed]
Areta, J.L.; Burke, L.M.; Ross, M.L.; Camera, D.M.; West, D.W.D.; Broad, E.M.; Jeacocke, N.A.; Moore, D.R.;
Stellingwerff, T.; Phillips, S.M.; et al. Timing and distribution of protein ingestion during prolonged recovery
from resistance exercise alters myofibrillar protein synthesis. J. Physiol. 2013, 591, 2319–2331. [CrossRef]
[PubMed]
Wagenmakers, A.J.M. Tracers to investigate protein and amino acid metabolism in human subjects.
P. Nutr. Soc. 1999, 58, 987–1000. [CrossRef]
Kelly, F.J.; Lewis, S.E.M.; Anderson, P.; Goldspink, D.F. Pre- and postnatal growth and protein turnover in
four muscles of the rat. Muscle Nerve 1984, 7, 235–242. [CrossRef] [PubMed]
Terjung, R.L. The turnover of cytochrome c in different skeletal-muscle fibre types of the rat. Biochem. J. 1979,
178, 569–574. [CrossRef] [PubMed]
Watt, P.W.; Kelly, F.J.; Goldspink, D.F.; Goldspink, G. Exercise-induced morphological and biochemical
changes in skeletal muscles of the rat. J. Appl. Physiol. 1982, 53, 1144–1152. [PubMed]
Burniston, J.G.; Hoffman, E.P. proteomic responses of skeletal and cardiac muscle to exercise. Proteomics
2011, 8, 361–377. [CrossRef] [PubMed]
Jaleel, A.; Short, K.R.; Asmann, Y.W.; Klaus, K.A.; Morse, D.M.; Ford, G.C.; Nair, K.S. In vivo measurement of
synthesis rate of individual skeletal muscle mitochondrial proteins. Am. J. Physiol. Endocrinol. Metab. 2008,
295, 1255–1268. [CrossRef] [PubMed]
Gasier, H.G.; Riechman, S.E.; Wiggs, M.P.; Previs, S.F.; Fluckey, J.D. A comparison of 2 H2 O and phenylalanine
flooding dose to investigate muscle protein synthesis with acute exercise in rats. Am. J. Physiol.
Endocrinol. Metab. 2009, 297, 252–259. [CrossRef] [PubMed]
Proteomes 2016, 4, 12
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
14 of 15
Gasier, H.G.; Fluckey, J.D.; Previs, S.F. The application of 2 H2 O to measure skeletal muscle protein synthesis.
Nutri. Metab. 2010, 7, 31–35. [CrossRef]
Gasier, H.G.; Fluckey, J.D.; Previs, S.F.; Wiggs, M.P.; Riechman, S.E. Acute resistance exercise augments
integrative myofibrillar protein synthesis. Metabolism 2012, 61, 153–156. [CrossRef] [PubMed]
Jones, P.J.; Leatherdale, S.T. Stable isotopes in clinical research: Safety reaffirmed. Clin. Sci. Lond. 1991, 80,
277–280. [CrossRef] [PubMed]
Busch, R.; Kim, Y.K.; Neese, R.A.; Schade-Serin, V.; Collins, M.; Awada, M.; Gardner, J.L.; Beysen, C.;
Marino, M.E.; Misell, L.M.; et al. Measurment of protein turnover rates by heavy water labeling of
nonessential amino acids. Biochim. Biophys. Acta 2006, 1760, 730–744. [CrossRef] [PubMed]
Xiao, G.G.; Garg, M.; Lim, S.; Wong, D.; Go, V.L.; Lee, W.P. Determination of protein synthesis in vivo using
labeling from deuterated water and analysis of MALDI-TOF spectrum. J. Appl. Physiol. 2008, 104, 828–836.
[CrossRef] [PubMed]
Jarvis, J.C.; Mokrusch, T.; Kwende, M.M.N.; Sutherland, H.; Salmons, S. Fast-to-slow transformation in
stimulated rat muscle. Muscle Nerve 1996, 19, 1469–1475. [CrossRef]
Burniston, J.G.; McLean, L.; Beynon, R.J.; Goldspink, D.F. Anabolic effects of a non-myotoxic dose of the
β2 -adrenergic receptor agonist clenbuterol on the rat plantaris muscle. Muscle Nerve 2007, 35, 217–223.
[CrossRef] [PubMed]
Burniston, J.G. Changes in the rat skeletal muscle proteome induced by moderate-intensity endurance
exercise. Biochim. Biophys. Acta 2008, 1784, 1077–1086. [CrossRef] [PubMed]
Burniston, J.G. Adaptation of the rat cardiac proteome in response to intensity-controlled endurance exercise.
Proteomics 2009, 9, 106–115. [CrossRef] [PubMed]
Hellerstein, M.K.; Neese, R.A. Mass isotopomer distribution analysis: A technique for measuring biosynthesis
and turnover of polymers. Am. J. Physiol. 1992, 263, 988–1001.
Commerford, S.L.; Carsten, A.L.; Cronkite, E.P. The distribution of tritium among the amino acids of proteins
obtained from mice exposed to tritiated water. Radiat. Res. 1983, 94, 151–155. [CrossRef] [PubMed]
Malik, Z.A.; Cobley, J.N.; Morton, J.P.; Close, G.L.; Edwards, B.J.; Koch, L.G.; Britton, S.L.; Burniston, J.G.
Label free LC-MS profiling of skeletal muscle reveals heart-type fatty acid binding protein as a candidate
biomarker of aerobic capacity. Proteomes 2013, 3, 290–308. [CrossRef] [PubMed]
Burnistion, J.G.; Meek, T.H.; Pandey, S.N.; Broitman-Maduro, G.; Maduro, M.F.; Bronikowski, A.M.;
Garland, T., Jr.; Chen, Y.W. Gene expression profiling of gastrocnemius of “minimuscle” mice.
Physiol. Genomics 2013, 45, 228–236. [CrossRef] [PubMed]
Kim, S.T.Y.; Wang, D.; Kim, A.K.; Lau, E.; Lin, A.J.; Liem, D.A.; Zhang, J.; Zong, N.C.; Lam, M.P.Y.; Ping, P.
Metabolic Labelling Reveals Proteome Dynamics of Mouse Mitochondria. Mol. Cell. Proteomics 2012, 11,
1586–1594. [CrossRef] [PubMed]
Burniston, J.G.; Connolly, J.; Kainulainen, H.; Britton, S.L.; Lauren, G.; Koch, L.G. Label-free profiling
of skeletal muscle using high-definition mass spectrometry. Proteomics 2014, 14, 2339–2344. [CrossRef]
[PubMed]
Rooyackers, O.E.; Balagopal, P.; Nair, K.S. Measurement of synthesis rates of specific muscle proteins using
needle biopsy samples. Muscle Nerve Suppl. 1997, 5, 93–96. [CrossRef]
Heilig, A.; Pette, D. Albumin in rabbit skeletal muscle. Origin, distribution and regulation of contractile
activity. Eur. J. Biochem. 1988, 171, 503–508. [CrossRef] [PubMed]
Donoghue, P.; Doran, P.; Dowling, P.; Ohlendieck, K. Differential expression of the fast skeletal muscle
proteome following chronic low-frequency stimulation. Biochim. Biophys. Acta 2005, 1752, 166–176.
[CrossRef] [PubMed]
Lewis, S.E.M.; Kelly, F.J.; Goldspink, D.F. Pre- and post-natal growth and protein turnover in smooth muscle
heart and slow- and fast-twitch skeletal muscles of the rat. Biochem. J. 1984, 217, 517–526. [CrossRef]
[PubMed]
Kenyani, J.; Medina-Aunon, A.; Martinez-Bartolomé, S.; Albar, J.P.; Wastling, J.M.; Jones, A.R. A DIGE study
on the effects of salbutamol on the rat muscle proteome-an exemplar of best practice for data sharing in
proteomics. BMC Res. Notes 2011, 86, 1756–1762. [CrossRef] [PubMed]
Proteomes 2016, 4, 12
31.
32.
15 of 15
Kasumov, T.; Dabkowski, E.R.; Shekar, K.C.; Li, L.; Ribeiro, R.F.J.; Walsh, K.; Previs, S.F.; Sadygov, R.G.;
Willard, B.; Stanley, W.C. Assessment of cardiac proteome dynamics with heavy water: Slower protein
synthesis rates in interfibrillar than subsarcolemmal mitochondria. Am. J. Physiol. Heart Circ. Physiol. 2013,
304, 1201–1214. [CrossRef] [PubMed]
Shankaran, M.; Shearer, T.W.; Stimpson, S.A.; Turner, S.M.; King, C.; Wong, P.A.; Shen, Y.; Turnbull, P.S.;
Kramer, F.; Clifton, L.; et al. Proteome-wide muscle protein fractional synthesis rates predict muscle mass
gain in response to a selective androgen receptor modulator in rats. Am. J. Endocrinol. Metab. 2015, 10,
1152–1201. [CrossRef] [PubMed]
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