Effect of Exercise on the Human Skeletal Muscle Phosphoproteome
Phase: NA
Conditions: Skeletal Muscle Protein Synthesis
Sponsor: McMaster University

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Protocol 
 
 
Study Title:  
Effect of exercise on the human skeletal muscle 
phosphoproteome 
 
Principal Investigator: 
 
Dr. Stuart Phillips1 
 
Physician overseeing medical responsibility:   
 
Dr. Steven Baker3 
 
Co-Investigators:   
 
 
Mr Jonathan Mcleod1  
 
Mr Aaron Thomas1 
 
Dr. Chris McGlory1  
 
Dr. Troy Hornberger2 
 
Institution: 
1 Department of Kinesiology, McMaster University, Hamilton, Ontario, L8S 4K1, 
Canada. 
2 Department of Comparative Bioscience, University of Wisconsin-Madison, 
Madison, Wisconsin, 53706, USA 
3 Department of Medicine, McMaster University, Hamilton, Ontario, L8S 4K1, 
Canada. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Rationale 
 
Skeletal muscle is a highly plastic tissue, capable of adapting to changes in 
nutritional intake and contractile activity. For instance, resistance exercise 
results in a mild stimulation of rates of muscle protein breakdown (MPB) but a 
greater stimulation of the rates of muscle protein synthesis (MPS) (1-3). When 
resistance exercise is performed prior to protein ingestion there is a synergistic 
combination of the two stimuli such that rates of MPS are stimulated over and 
above those of MPB (4-6). Thus, repeated bouts of resistance exercise, when 
coupled with protein ingestion, result in the accretion of skeletal muscle protein 
referred to as hypertrophy (7). Importantly, by changing the nature of the 
exercise stimulus, it is possible to redirect the focus of the type of skeletal 
muscle proteins that are being synthesised. For example, we know that 
prolonged and repeated lower-load dynamic stimulation of skeletal muscle (i.e., 
endurance exercise training) results in an increase in the expression of 
mitochondrial genes (8), proteins (8-10), and ultimately enhanced mitochondrial 
content (11), leading to a shift towards an oxidative phenotype, and improved 
fatigue resistance (12). Resistance exercise training also stimulates the 
transcription of genes and accrual of new muscle proteins (13, 14), but these 
genes and proteins are largely associated with the myofibrillar protein fraction, 
and regular resistance exercise leads to muscle hypertrophy and increased 
force-generating capacity (15, 16). However, during the early stages of exercise 
training, particularly in training-naïve participants there is a significant increase 
in the expression of genes common to both modalities of exercise (17, 18). It is 
only with sustained exercise training that there is a ‘fine tuning’ of the 
transcriptome, the protein synthetic response, and then the proteome that gives 
rise to divergent hypertrophic and oxidative phenotypes (17, 19).   
 
 
Transcriptional responses to exercise training 
 
Increases in mRNA expression following each exercise session result in 
enhanced translation of proteins, and ultimately adaptive changes in muscle 
protein content (12, 19). The temporal pattern of these changes with respect to 
endurance and resistance exercise training is now becoming clearer. Exercise-
induced mitochondrial biogenesis, for example, is a characteristic feature of 
endurance exercise training (9, 12, 20) and is underpinned by the coordinated 
up-regulation of both mitochondrial and nuclear transcripts that encode for 
proteins involved in the electron transport chain (21-23), and lipid metabolism 
(24). These transcripts include peroxisome proliferator-activated receptor 
gamma co-activator (PGC)-1α, nuclear respiratory factors (NRFs), and the 
mitochondrial transcription factor A (TFAM)(25-27). An acute bout of endurance 
exercise activates sensors of cellular stress (8, 28), decreases methylation of 
promoter regions (29), stimulates phosphorylation of mediators of translation 
initiation (16), and increases the expression of the aforementioned transcripts 
(PGC-1α, NRFs, TFAM) (8, 12). The transient increase in the expression of 
genes in the hours following an acute endurance exercise session provides the 
gene template that precedes the respective increases in protein content 
observed during exercise training (8, 9). Compared to our understanding of 
transcriptional responses to endurance training, the corresponding picture with 

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respect to resistance exercise is relatively less complete. Resistance exercise 
results in the phosphorylation (i.e., activation) of the mechanistic target of 
rapamycin complex 1 (mTORC1)(30-33). mTORC1 activation serves to 
enhance MPS by (1) activating downstream protein kinases such as the 
ribosomal protein of 70 kDa S6 kinase 1 (p70S6K1) and 4E-binding protein-1 
(4EBP1), which subsequently promote ribosomal binding to mRNA to initiate 
protein synthesis (34-37) and (2) up-regulating the transcription of the 
translational machinery itself (mRNA, ribosomal content) (38). Thus, resistance 
exercise stimulates an increase in mTORC1 activity and promotes increases in 
the rates of myofibrillar MPS through both increased translational efficiency 
(protein synthesized per unit of mRNA) and translational capacity (the 
abundance of the translational machinery; ribosomes) (31, 33).  
 
 
Posttranslational modification of proteins 
 
Key to exercise-induced adaptation is post-translational modification of 
proteins. One important post-translational modification is known as 
phosphorylation. Phosphorylation is the addition of a phosphate group to a 
target protein by a protein kinase using ATP and Mg (39). The addition of this 
phosphate can promote the catalytic activity or binding status of a protein 
resulting in a biological downstream effect. There are over 500 kinases in the 
human ‘kinome’. We (39, 40) and others (16) often assess the phosphorylation 
status of single or multiple (~ten) kinases/proteins in response to exercise to 
gain a mechanistic understanding of how exercise alters the skeletal muscle 
phenotype. However, current methods are largely semi-quantitative (Western 
blotting) and are biased in terms of which proteins are studied (i.e., the 
experimenter can only select a limited number of kinases/proteins to measure). 
Our understanding of how skeletal muscle adaptation is influenced by 
phosphorylation is therefore limited. Recent developments in mass 
spectrometry (41, 42) have enabled a wider interrogation of the phosphorylation 
status of proteins in response to exercise. Indeed, one study, using advanced 
liquid chromatography-mass spectrometry, revealed 1,004 unique exercise-
regulated phosphorylation sites on 562 proteins in human skeletal muscle 
biopsy samples obtained before and after a bout of endurance exercise (41). 
This seminal finding has significantly advanced our knowledge regarding how 
endurance exercise impacts phosphorylation. Yet, no measurements of end-
point MPS were made. Moreover, comparable work has not been performed 
with resistance exercise. The aim of our investigation is to conduct a similar 
global phosphoproteomic analysis of skeletal muscle using advanced mass 
spectrometry following both endurance and resistance exercise in humans. We 
will also directly measure rates of MPS for the 3 hours following exercise.  
 
The aim of our investigation is to conduct a similar global phosphoproteomic 
analysis of skeletal muscle using advanced mass spectrometry following 
resistance and aerobic exercise in humans.  

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Specific Objectives 
 
1. Our primary objective is to examine the effect of  resistance exercise on the 
human skeletal muscle phosphoproteome. 
 
2. Our secondary objective is to assess changes in rates of myofibrillar and 
mitochondrial MPS following resistance and endurance exercise.  
 
 
 
Hypotheses 
 
We hypothesize that: 
 
1. Both resistance exercise and endurance exercise will result in the 
widespread phosphorylation of proteins involved in the regulation of energy 
metabolism, transcription, translation and muscle protein synthesis. 
 
2. Resistance exercise will increase myofibrillar MPS to a greater extent than 
endurance exercise. 
 
3. Endurance exercise will increase rates of mitochondrial MPS to a greater 
extent than resistance exercise.  
 
Participants 
 
Inclusion Criteria 
In order to participate in this study, participants must be male between the ages 
of 18 and 30 years and have performed resistance exercise 3 times per week 
for at least 1 year.  
 
Exclusion Criteria 
Participants cannot participate in, or will be released from, this study if they 
meet one of more of the following criteria:  
 
• Smoker or user of tobacco products 
• High physical activity. 
• Have health problems such as: renal or gastrointestinal disorders, metabolic 
disease, heart disease, vascular disease, rheumatoid arthritis, diabetes, 
poor lung function, uncontrolled blood pressure, dizziness, thyroid 
problems, or any other health conditions for which you are being treated that 
might put you at risk for this study. 
• Taking anti-diabetic, anti-inflammatory, platelet inhibitor, or anti-coagulant 
medications. 
• Use of an investigational drug product within the last 30 days. 
• Have participated in an infusion protocol in the last year. 
• Do not understand English or have a condition the PI believes would 
interfere with a participants’ ability to provide informed consent, comply with 

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the study protocol, or which might confound the interpretation of the study 
results or put someone at undue risk. 
 
 
Recruitment 
 
Twelve participants (6 male: 6 female) participants will be recruited for this 
study through poster advertisements. Potential participants will be asked if they 
meet all of the inclusion criteria and none of the exclusion criteria. During an 
initial screening visit those who qualify and are interested in participating will be 
invited to the Exercise Metabolism Research Group (EMRG) laboratory, Ivor 
Wynne Centre (IWC) at McMaster University to begin testing for the study. The 
potential participants will then be required to read the Information and Consent 
Form. A written copy of the consent form will be provided for the participants to 
read by themselves. Written informed consent will then be obtained.  
 
Sample Size 
 
This study is an observational pilot investigation. Previous work has detected 
changes in our primary outcome (phosphoproteome) following endurance 
exercise with only 4 male participants (41). For the current study, we wish to 
recruit both male and female participants. Therefore - accounting for participant 
attrition - we will aim to recruit 12 participants. 
 
Methodology 
 
A schematic illustration of the experimental design and experimental trial can 
be seen in Figures 1 and 2, respectively. In a within-subject repeated measures 
cross-over design, 12 participants (6 male and 6 female) will attend the 
laboratories at the Dept. of Kinesiology on 6 separate occasions. On the first 
occasions participants will be assessed for body composition using dual-energy 
x-ray absorptiometry (DXA) after an overnight fast (~10 hours) followed by 
familiarization on resistance exercise and endurance exercise equipment. At 
this point, your two legs will be randomly assigned to the resistance exercise or 
endurance exercise group. 
 
On the second visit, participants will perform a 1-repetition maximum test on a 
leg press and leg extension machine. These tests will be used to determine the 
workload performed on the experimental trials. On the third visit participants will 
perform a V02 max test on a cycle ergometer to determine their aerobic fitness. 
On the same day, subjects will be familiarized with single-leg cycling. On visit 
4, subjects will perform another V02max test on a cycle ergometer using one leg 
only. This test will determine the intensity of cycling performed on the 
experimental trial. To ensure the intensity is appropriate, the subjects will be 
familiarized with the experimental trial training programs (see “Exercise 
protocols” below). 
 
Subjects will then embark on a 3-day period of controlled diet and exercise 
followed by experimental trials (visits 5 and 6) separated by 1 week. The reason 
for the two independent trails is that the measurement of MPS requires the 

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infusion of a stable amino acid isotope (L-[ring-13C6] phenylalanine), which 
interferes with the assessment of the phosphoproteome due to the fact they 
both detect changes in protein mass. Thus the first trial will be conducted to 
assess exercise-induced changes in the phosphoproteome and the second for 
MPS, using the L-[ring-13C6] phenylalanine isotope.  
 
 
Acute protocols (Visits 5 + 6) 
 
On visit 5 participants will arrive at the laboratory at approximately 0640 after a 
10h overnight fast. After a brief assessment, subjects will rest on a bed for 3 
hours. After this period, a skeletal muscle biopsy will be obtained and 
participants will perform a bout of resistance exercise with a randomised leg, 
followed by a bout of endurance exercise. Skeletal muscle biopsies will again 
be obtained immediately following both bouts of exercise and again 3h following 
exercise. On visit 6, participants will again arrive at the laboratory at 
approximately 0640 after an overnight fast. This time, a catheter will be placed 
in the upper forearm. After the placement of the catheter, a primed constant 
infusion of L-[ring-13C6] phenylalanine will be commenced to measure rates of 
MPS and a catheter placed in the contralateral arm for repeated blood 
sampling. Exercise and skeletal muscle biopsies will be obtained as in visit 5 
(see Figure 2). We have routinely obtained ethical approval and published 
papers for studies that employ skeletal muscle biopsies and stable amino acid 
tracers in human volunteers (3, 14, 44-49). Dr. Steven Baker, a liscenced 
physician, has approved our standard operating procedure and will oversee all 
muscle biopsy and catheter infusions.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Figure 1 Schematic illustration of experimental design.  
 
 
 
 
 
 
Figure 2. Schematic illustration of Visit 6. 
Familiarization
DXA 
1 RM testing
Double-leg 
VO2 max &
Single-leg
familiarization
Single-leg
VO2 max
& practice
3 d controlled 
exercise and diet
Experimental 
trial
0d                             3d                              5d                             7d                         
11d                                        14d
Week 0
Week 1
Week 2 
Experimental trial 2
Week 3
21d
.
.
-200            -180          -60                           0        20                     60                           120    
180       200              240
6:40am      7am        9am                      10am                         11am                       12pm                 
1pm                         2pm
Primed constant infusion of L-[ring-13C6] phenylalanine and repeated blood sampling
R.Ex 
bx
E.Ex 
bx
E.Ex
R.Ex
R.Ex 
bx
E.Ex 
bx
Pre 
bx
Arrival
* Timing will change depending on exercise order
Bed rest
Bed rest
R.Ex- 3 sets × 10 reps 70% 1RM leg press followed by leg ext. 
2 min recovery between sets and exercises. 
E.Ex- 4 × 5 min @ 65% single leg peak power
w/ 2.5 min recovery @ 20% peak power. 

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Measurementsand Measurement Instruments 
 
1 repetition maximum: 
The same investigators will administer all strength testing. In short, after a 
brief general warm-up, a specific warm-up of the given exercise will be 
performed at approximately 50% of the participant’s estimated 1RM based on 
the 10RM testing. Load will be progressively increased by approximately 10-
20% for each repetition until a true 1RM will be reached as previously 
described (39). Three to 5 min of rest was given between each attempt. A 
successful attempt required the participant to move the load throughout the 
full range of motion with correct form. 
 
VO2peak  
Subjects will complete double-leg and single-leg incremental peak oxygen 
uptake (VO2peak) tests on a cycle ergometer (Velotron, RacerMate; Seattle, 
WA). A metabolic cart with an on-line gas collection system (Moxus modular 
oxygen uptake system, AEI Technologies, Pittsburgh, PA) will acquire oxygen 
consumption (VO2) and carbon dioxide production (VCO2) data and heart rate 
will be monitored continuously with a heart rate monitor (Polar A3, Lake 
Success, NY). The test will begin with a 1-min warm up at 50 watts (W), after 
which the power will be increased by 1 W every two seconds until volitional 
exhaustion or the point at which pedal cadence falls below 60 rpm. Heart rate 
will be monitored continuously throughout the test via telemetry with a heart 
rate monitor (Polar, Kempele, Finland). A similar protocol will be used for single-
leg tests, except that power will increase by 1 W every four seconds. VO2peak 
and will be defined as the highest oxygen consumption achieved over a 30-s 
period. Maximal workload (Wmax) will be the highest power output achieved 
during the test. The subject is deemed to have reached VO2peak if: i) the 
perceived exertion is >19 (Borg Scale) ii) his HR is within 5bpm of age-predicted 
maximal HR; iii) his RER is >1.2; and iv) a plateau has been reached in their 
oxygen consumption. 
 
DXA Scan:  
Body composition (i.e. fat (%), appendicular lean mass (kg), and lean body 
mass (kg)) will be determined by dual-energy x-ray absorptiometry (DXA). 
 
Deuterated Water:  
Deuterium is an isotope of hydrogen and is in fact already present in small 
amounts in the water consumed daily (i.e. 0.02%). It poses no health risk in 
the quantities consumed during this study, and about half of the water in the 
body is replaced every week. Once the participant stops drinking this water, 
body water will return to its normal concentration in 20-30 days. During the 
study, participants will be required to drink ~1 cup of deuterated water (D2O). 
 
Skeletal Muscle Biopsies and Analyses: 
During the study, 7 muscle biopsies will be collected (days -3, 0 [4 biopsies], 1, 
and 2). This procedure involves the removal of a small piece of muscle tissue 
using a sterile hollow needle from the quadriceps muscle (vastus lateralis) 
under local aesthetic (lidocaine). A small piece of muscle (~100 mg) will be 

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collected at each time point. Following the biopsies, the incisions will be closed 
with instant medical adhesive, and wrapped with a tensor bandage. 
Phosphoproteomic analysis will be conducted by mass-spectrometry as 
previously described (41). Myofibrillar MPS will be calculated using the 
precursor-product equation: myofibrillar MPS =([E2b-E1b]/[Eic x t]) x 100. Eb 
represents the enrichment of bound myofibrillar protein, Eic is the average 
intracellular enrichment between two biopsies, and t is the tracer incorporation 
time in h. As we will employ ‘tracer naïve’ participants (had not previously 
participated in a study protocol where L-[ring- 13C6] phenylalanine was infused), 
a pre-infusion blood sample will be used for the calculation of resting myofibrillar 
MPS (50). Myofibrillar and intracellular enrichments of L-[ring- 
13C6] 
phenylalanine will be measured as previously described (51). Integrated (i.e., 
days) rates of muscle protein synthesis (MPS) will be determined using the D2O 
method. Briefly, 2H enrichment of saliva (precursor) and muscle (product) pools 
(relative to 1H) will be determined using isotope ratio mass spectrometry and 
myofibrillar fractional synthetic rate determined, as previously described (52, 
53). 
 
Diets: 
Ad libitum 3-day dietary records for each participant will be collected and 
analyzed for baseline macronutrient intake. During the study, all food will be 
provided to the participants as frozen meals (Supplier: Copper County Foods), 
and pre-weighed and measured snack items.  
 
Physical activity: 
Recreationally active young men (assessed through physical activity 
questionnaire) will be recruited. Participants will be asked to maintain their 
habitual activity for the duration of the study. 
 
Exercise Protocol: 
Resistance exercise: 5 sets × 10 reps 75% 1RM leg press followed by leg 
extension, with 2 min recovery between sets and exercises (50).  
Endurance exercise: 4 × 5 min @ 65% single leg peak power with 2.5 min 
recovery @ 20% peak power (MacInnis 2016)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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