Development of an UPLC-mass spectrometry method for 1

measurement of myofibrillar protein synthesis: application to 2

analysis of murine muscles during cancer cachexia 3

Maria Lima1, Shuichi Sato2, Reilly T. Enos2, John W. Baynes3 and James A. Carson2* 4

1 Research and Innovation Centre, Fondaizone Edmund Mach – IASMA, San Michele all’ Adige 5

Trento, Italy 6 2 Department of Exercise Science, University of South Carolina, Columbia, South Carolina, 7

United States of America 8 3 Department of Pharmacology, Physiology and Neuroscience, University of South Carolina 9

School of Medicine, Columbia, SC, United States of America 10

* Correspondence to: 11

J. A. Carson, Dept. of Exercise Science, Public Health Research Center, Univ. of South Carolina, 12

921 Assembly St., Rm 405, Columbia, SC 29208 (email: carsonj@sc.edu) 13

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Running Head: Myofibrillar protein synthesis in skeletal muscle 15

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Keywords: cachexia, myofibrillar protein synthesis, soleus, plantaris, ultra performance liquid 17

chromatography-mass spectrometry 18

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Articles in PresS. J Appl Physiol (January 17, 2013). doi:10.1152/japplphysiol.01141.2012

Copyright © 2013 by the American Physiological Society.

ABSTRACT 23

Cachexia, characterized by skeletal muscle mass loss, is a major contributory factor to patient 24

morbidity and mortality during cancer. However, there are no reports on the rate of myofibrillar 25

protein synthesis (MPS) in skeletal muscles that vary in primary metabolic phenotype during 26

cachexia, in large part because of the small-size muscles and regional differences in larger 27

muscles in the mouse. Here we describe a sensitive method for measurement of MPS and its 28

application to analysis of MPS in specific muscles of mice with (ApcMin/+) and without 29

(C57BL/6) cancer cachexia. Mice were injected with a loading dose of deuterated phenylalanine 30

(D5F) and myofibrillar proteins extracted from skeletal muscles at 30 min. The relative 31

concentrations of D5F and naturally occurring phenylalanine (F) in the myofibrillar proteins and 32

the amino acid pool were quantified by ultra-performance liquid chromatograph (UPLC)-mass 33

spectrometry (MS). The rate of MPS was determined from D5F: F ratio in the protein fraction, 34

compared to the amino acid pool. The rate of MPS, measured in 2-5 mg muscle protein, was 35

reduced by up to 65% with cachexia in the soleus, plantaris, diaphragm, and oxidative and 36

glycolytic regions of the gastrocnemius. The rate of MPS was significantly higher in the 37

oxidative vs. glycolytic gastrocnemius muscle. A sufficiently sensitive UPLC-MS method 38

requiring very small amount of muscle has been developed to measure the rate of MPS in various 39

mouse muscles. This method should be useful for studies in other animal models for quantifying 40

effects of cancer and anti-cancer therapies on protein synthesis in cachexia, and particularly for 41

analysis of sequential muscle biopsies in a wide range of animal and human studies. 42

43

INTRODUCTION 44

Skeletal muscle is a highly plastic tissue, which can undergo hypertrophy or atrophy in response 45

to a variety of stimuli, including: disuse, variations in nutritional and hormonal status, aging, 46

exercise, chronic diseases and associated metabolic syndromes (15, 21, 27). Cachexia, is a 47

chronic wasting condition associated with inflammatory diseases such as cancer and is 48

characterized by severe loss of both muscle and adipose tissue mass (11, 20). Losses in muscle 49

mass lead to muscle weakness and impaired muscle function, which can negatively impact 50

quality of life, response to therapy, and can increase mortality (22, 24, 26). Cachexia affects up 51

to 80% of patients with advanced cancers and more than 20% of patients die as a result of 52

cachexia-related complications (1, 2, 29). The rapid progressive muscle loss rooted in cachexia is 53

the result of an imbalance between decreased protein synthesis (MPS) and increased protein 54

degradation (28). Because of its importance as a marker of cachexia, measurement of MPS is a 55

valuable tool for evaluating the effectiveness of strategies to combat cachexia. 56

Although there are several different animal models that are used to study cachexia, the 57

genetically-engineered ApcMin/+ mouse model (Min: multiple intestinal neoplasia) is commonly 58

utilized; the slow-progressive cachexia and associated side effects exhibited by this model 59

closely mimic human cachexia (4, 6). The ApcMin/+ mouse is heterozygous for a point mutation in 60

the adenomatous polyposis coli (Apc) gene, which leads to development of familial adenomatous 61

polyposis (FAP), a form of colon cancer. The mouse develops intestinal polyps at approximately 62

4 weeks of age, and loses body weight gradually between 14 and 20 weeks of age. At 20 weeks 63

of age, the mouse is usually severely cachectic, having lost more than 15% of body mass 64

compared to its peak body mass (4, 6, 25). Muscle phenotype related to the capacity for 65

oxidative metabolism can affect myofiber susceptibility to wasting (17), and primarily glycolytic 66

myofibers in the ApcMin/+ mouse demonstrated more atrophy during the development of cachexia 67

(5). The standard procedure for measuring protein synthesis in vivo involves measurement of 68

incorporation of isotopically labeled tracers into myofibrillar protein, using liquid scintillation 69

counting, isotope ratio mass spectrometry (IRMS), and gas chromatography/mass spectrometry 70

(GC-MS) (16, 23, 31). Although these methods have been useful, they require relatively large 71

amounts of tissue (typically 20 - 50 mg wet weight), which in some cases is not readily available, 72

except by pooling samples, and in any case, limits the availability of tissue needed for other 73

analyses (e.g. western blots, RT-PCR, etc) (14, 23). To date, many researchers have been 74

utilizing various mouse models of cachexia to understand the mechanisms responsible for muscle 75

wasting, and the role of muscle phenotype in the process (7, 9, 12, 18). 76

Tandem Mass spectrometry (MS/MS), coupled with ultra-performance liquid 77

chromatography (UPLC), provides high resolution, specificity and sensitivity for measurement 78

of amino acids in tissues (8). In this paper we describe a method for analysis of MPS in 79

individual mouse muscles using UPLC-MS/MS. 80

81

METHODS 82

Materials 83

Nonafluoropentanoic acid (NFPA, 97%) and sodium hydroxide (NaOH) were purchased 84

from Sigma (St. Louis, MO). Trifluoroacetic acid (TFA) was purchased from ThermoFisher 85

Scientific (Waltham, MA). L-[ring-2H5]phenylalanine (D5F) was from Cambridge Isotope 86

Laboratories (Andover, MA), and HPLC-grade water and methanol from VWR, Inc. (Radnor, 87

PA). 88

Animals & Tissue Collection 89

Male ApcMin/+ mice on a C57BL/6 background were originally purchased from Jackson 90

Laboratories (Bar Harbor, ME) and crossed with C57BL/6 female mice at the University of 91

South Carolina’s animal resource facility. Offspring were genotyped as heterozygotes by RT-92

PCR for the Apc gene as described by Mehl et al.(18, 19) All mice were housed in standard 93

cages. The room was maintained at 24 °C on a 12:12 light:dark cycle with the light period 94

starting at 0700. All mice were provided with standard rodent chow (Harlan Teklad Rodent Diet, 95

#8604, Madison, WI) and water ad libitum. Male C57BL/6 and ApcMin/+ mice (n = 6-7) were 96

sacrificed at 20 weeks of age. 97

Thirty minutes prior to sacrifice, all mice received an intraperitoneal injection of 150 mM 98

D5F in a 75 mM NaCl solution at a dose of 0.02 mL/g body weight. Subsequently, mice were 99

given a subcutaneous injection of a ketamine-xylazine-acepromazine cocktail (1.4 mL/kg body 100

weight). Muscles (plantaris, soleus, gastrocnemius, and diaphragm) were excised under 101

anesthesia, rinsed in PBS, weighed, snap frozen in liquid nitrogen, and stored at -80 °C until 102

further analysis. Red and white fibers were isolated from the gastrocnemius as previously 103

described (32). All animal experimentation was conducted in accordance with procedures 104

approved by the University of South Carolina’s Institutional Animal Care and Use Committee. 105

106

Protein Extraction and Isolation 107

108

Frozen muscle tissues were processed using the method of Welle et al (31) for 109

measurement of myofibrillar protein synthesis by gas chromatography mass spectrometry. Tissue 110

samples (~5 mg) were weighed in a 1.5 mL Eppendorf plastic centrifuge tube, to which 200 µL 111

of ice cold homogenizing buffer (HB: 50 mM KPO4, pH 7.0, 0.25 M sucrose, 1% Triton X-100) 112

was added, and kept on ice. Each sample was sonicated twice for 20 seconds on ice. The 113

sonicate was centrifuged at 10,000 g for 10 min at 4 °C, and the supernatant containing the free 114

amino acid pool was transferred to a fresh tube and dried under vacuum (Speed Vac, Thermo 115

Electron Corp., Milford, MA), then reconstituted in 1% aqueous TFA (1 mL) and stored at -116

20 °C. The pellet fraction was resuspended by sonication in 350 μL of HB, and centrifuged at 117

10,000 g for 10 min at 4 °C. The supernatant was discarded, and this washing procedure was 118

repeated once with HB (350 uL) and twice with ice-cold deionized water (500 uL). 119

NaOH (0.3 M, 200 uL) was added to the final pellet followed by centrifugation at 10,000 120

g for 10 min at 4 °C. The supernatant, containing myofibrillar proteins, was diluted with 6 M 121

HCl (1 mL) and the protein hydrolyzed at 110 °C for 24 h in 2 mL screw-closure polypropylene 122

O-ring sealed vials (Sarstedt, Nümbrecht, Germany). HCl was removed under vacuum (Speed 123

Vac, Thermo Electron Cooperation, Milford, MA), and the dried protein hydrolysate was 124

reconstituted in 1% aqueous TFA (1 mL) and stored at -20 °C. The free amino acid pool and 125

myofibrillar protein hydrolysate were applied to a 3 mL Supelco C18 cartridge (Waters, Milford, 126

MA), equilibrated in 1% aqueous TFA. The aromatic amino acid fraction was eluted with 1% 127

TFA in methanol:water (20:80, v/v, 3 mL). The eluate was dried under vacuum and reconstituted 128

in aqueous 5 mM NFPA (45 μL) prior to analysis by UPLC-MS/MS. 129

130

LC-MS Conditions and Instrumentation 131

132

Samples were fractioned on a Waters (Manchester, UK) Acquity UPLC-MS/MS system using a 133

BEH C18 column (2.1 x 50 mm, particle size 1.7 µm), which was maintained at room 134

temperature. Samples were eluted at a flow rate of 0.2 mL/min, eluting for 2.0 min with 100% 135

solvent A (aqueous 5 mM NFPA), followed linear gradient to 50% solvent B (acetonitrile) over 136

10 min, then washed with 80% solvent B for 15 min. The injection volume was 5 µL (equivalent 137

to ~ 3 nmol of F). 138

Mass spectrometry experiments and optimization of the method were performed utilizing 139

a Micromass Quattro Premier XE Tandem Mass Spectrometer (Waters). Multiple reaction 140

monitoring (MRM) was conducted operating the MS in positive ion electrospray mode. Table 1 141

details the MRM conditions of analysis. Other MS operating conditions used were: scan 142

duration, 1s; capillary voltage, 3 kV; cone voltage, 25 eV; source temperature, 100 °C; 143

desolvation temperature, 350 °C. Sample peaks corresponding to phenylalanine, naturally 144

occurring 13C-phenylalanine and D5F were integrated. Data were analyzed using MassLynx 145

software (version 4.1), supplied by Waters. Analytes were quantified by comparing the peak 146

area of deuterated compound: unlabelled compound. Percent protein synthesis per day was 147

calculated, using the general formula (31) by dividing the ratio of D5F/F in myofibrillar protein 148

pellet by the ratio of D5F/F in the free amino acid pool and expressing it as a percentage of newly 149

synthesized protein, using the following formula: 150

151

152

153

154

155

Statistical Analysis 156

157

%MPS /day =

D5FF (MPP)

D5FF (FAAP)

×100 × 24h

0.5h

MPS- myofibrillar protein synthesis, MPP- myofibrillar protein pellet, FAAP- free amino acid pool

All statistical analyses were performed using GraphPad Prism (La Jolla, CA). All 158

comparisons were analyzed using a Student’s two-tailed t-test, with significance set at p ≤ 0.05. 159

160

RESULTS AND DISCUSSION 161

UPLC-MS conditions were optimized to resolve phenylalanine in a short 162

chromatographic run. Addition of 5 mM NFPA, an ion-pairing agent, to the mobile phases 163

improved signal intensity and resolution. MRM conditions were manually optimized for each 164

compound in positive ESI ionization mode to obtain maximal response. The mass spectrum of 165

phenylalanine is shown in Fig. 1A. The precursor ion (m/z 166) was selected for fragmentation 166

and the fragment corresponding to the immonium ion (m/z 120), which was the most abundant 167

fragment in the spectrum, was recorded by the second mass analyzer. Chromatograms obtained 168

for F, 13C-F and D5F are shown in Fig. 1B and 1C for myofibrillar protein pellet and the amino 169

acid pool, respectively. The natural abundance peak of 13C-F, which is 1.1 % the intensity of the 170

12C-F peak, was used for internal standardization since the 12C-F signal saturated the detector for 171

some samples. 172

All peaks were normalized to the strongest signal in the pellet and amino acid pool 173

fractions, respectively. As expected, the myofibrillar pellet contained an abundant amount of F, 174

followed by naturally occurring 13C-F, which accounts for ~ 10% of F, and D5F. Although the 175

peak intensity for the D5F in pellet was only ∼ 0.14% of F in the myofibrillar protein, this was 176

well within the linear range of the instrument and yielded a strong signal during MRM analysis 177

(Fig. 1B, insert). 178

Using the optimized MRM conditions and the formula above, myofibrillar protein 179

synthesis was quantified in various skeletal muscles (Fig. 2). The mice chosen for the study, at 180

20 weeks of age, were chosen from a group of severely cachectic Apc Min/+mice (Table 2). 181

Cachectic Apc Min/+mice experience an average of ~20% weight loss between their 12th and 19th 182

week of age (32). Not all of the weight changes the result of loss of skeletal muscle - there are 183

comparable losses of adipose tissue mass. Even with adjustment for the lifespan of mice, 184

compared to humans, the rate of weight loss in untreated severely cachectic mice is extreme. In 185

humans, pre-cachexia is generally defined as ≤5% weight loss during a 6-month period, while 186

weight loss ≥6% during this period is described as cachexia (3). 187

The rate of MPS varied from ~1.25 %/d to ~2.5 %/d in white and red gastrocnemius, 188

respectively, to ~4.3 %/day in the diaphragm muscle of control animals at 20 weeks (5 months) 189

of age (Fig. 2), in reasonable agreement with the 3% estimate of MPS in the gastrocnemius of 190

C57BL/6 mice at 6 months of age, analyzed by the GC-MS/MS procedure (31). MPS was 191

significantly reduced in all muscles in cachectic animals, but varied significantly with the 192

specific muscle, from ~60% in the red gastrocnemius and diaphragm muscles, to ~75% in the 193

plantaris muscle (Fig. 2). In a previous study, using a GC-MS/MS procedure, we observed an 194

~50% decrease in the rate of gastrocnemius muscle protein synthesis in a group of animals with 195

≥20% weight loss (33), which is reasonably consistent with the results of the UPLC-MS/MS 196

method applied to a more severely cachectic group of mice. There is also an ~150% increase in 197

the rate of muscle protein degradation in these animals (33), which further contributes to the 198

decrease in muscle mass. Percentage reductions in MPS were observed across all muscle types, 199

including the soleus and plantaris muscles, which could not be measured (without pooling 200

samples) by the GC-MS/MS method. We also show for the first time that the absolute rate of 201

MPS is significantly higher in the red portion of the gastrocnemius and the soleus muscle 202

compared to the white portion of the gastrocnemius and the plantaris muscle in the mouse. This 203

is consistent with the observation that MPS in glycolytic muscle fibers is more susceptible to 204

wasting compared to oxidative fibers in humans and rats (10, 13, 30). However, to the best of our 205

knowledge, no other studies have examined the rate of MPS in different muscle phenotypes of 206

cachectic mice due to limitations regarding tissue mass needed for the analysis. 207

To further validate the method, MPS was measured in younger ApcMin/+mice group 208

compared to the C57BL/6 controls animals (aged 10 wk) (Fig. 3). These results illustrated that 209

there was no significant difference in MPS across genotypes, which supports the observation that 210

Apc Min/+mice are not cachetic at this age (33), hence MPS is not decreased. Compared to the 211

GC-MS/MS method (33), which requires ~ 50 mg of tissue per analysis, the current method 212

requires only 5 mg of tissue, which permits triplicate injections and applies more straightforward 213

calculations. The intra-assay variance within a group of animals was < 2. Limit of detection is ~ 214

50 fmol for F standard injected on the column. Compared to GCMS, which requires 215

derivatization and longer analysis time, this method is far more advantageous due to its 216

simplicity of sample preparation and short analysis time. The small sample requirement for the 217

UPLC-MS/MS assay also allows for analysis of MPS in subcellular fractions of mouse muscles, 218

and should also be applicable to the analysis of sequential biopsy samples from animal and 219

human studies. 220

In summary, a specific, sensitive, and relatively simple method has been developed to 221

measure MPS in mouse tissues and has been applied to analysis of MPS in control and cachectic 222

mice. This method has been applied to analysis of small muscles (soleus and plantaris) muscles 223

and to analysis of MPS in different regions of the gastrocnemius muscle (red vs. white) 224

of ApcMin/+ mice. These results show that the rate of MPS is greater in red, oxidative muscles, 225

and that the rate of MPS is comparably decreased in all of these muscles including the 226

involuntary skeletal muscle diaphragm in ApcMin/+ mice during cachexia. Future studies on the 227

rates of protein turnover in these tissues will provide a better understanding of the relative roles 228

of changes in muscle protein synthesis vs. turnover in the loss of muscle mass during cachexia. 229

230

ACKNOWLEDGEMENTS 231

The authors thank Dr. William Cotham for technical assistance with mass spectrometry 232

analyses, and Drs. James A. White and Norma Frizzell for helpful discussions. 233

234

GRANTS 235

This work was supported by the grant R01CA121249. 236

237

DISCLOSURES 238

The authors have declared that they have no competing interests. 239

240

AUTHOR CONTRIBUTIONS 241

Conceived and designed the experiments: ML SS RTE JWB JAC. Performed the 242

experiments: ML SS RTE JAC. Analyzed the data: ML SS RTE JWB JAC. Contributed 243

reagents/materials/analysis tools: ML JWB JAC. Wrote the paper: ML SS RTE JWB JAC. 244

245

246

REFERENCES 247

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Table 1. MRM condition of analysis

Compound Parent Mass (Da)

Daughter Mass (Da)

Cone Voltage

Collision energy (eV)

D5-F 171 125 25 13 C13-F 167 121 25 13

F 166 120 25 13

Table 2. Body weights and muscle weights of C57BL/6 and ApcMin/+ mice

C57BL/6 ApcMin/+ N 7 6 Body weight (g) 29.2 ± 1.3 18.6 ± 0.7 * Soleus (mg) 10.0 ± 0.6 6.3 ± 0.5 * Plantaris (mg) 18.1 ± 0.6 10.5 ± 0.8 * Gastrocnemius (mg) 134.3 ± 3.5 68.5 ± 6.1 * Diaphragm 71.3 ± 1.6 30.5 ± 2.7 *

* Significantly different from age-matched C57BL/6 control mice (p <0.05)

Figure 1. Optimization of mass spectrometric conditions for analysis of phenylalanine. Fragmentation pattern of phenylalanine ions (A), extracted ion chromatograms for phenylalanine (F), naturally occurring Carbon 13 F (C13 F), and Deuterated F (D5F) incorporated in the myofibrillar protein pellet (B) (D5F was magnified 100 X, insert shows the well resolved chromatogram for D5F without magnification) and supernatant (C). All peaks are normalized to the largest occurring peak either in the pellet or supernatant fraction. Figure 2. Skeletal muscle protein synthesis is decreased during cancer cachexia. Myofibrillar protein synthesis was measured at 20 weeks of age in gastrocnemius (white, glycolytic portion) (A) and gastrocnemius (red, oxidative portion) (B) plantaris (C) soleus (D) and diaphragm (E) muscle. Values are mean ± SE, n ≥ 5. *Signifies difference from wild type (p < .05) #Signifies difference from white gastrocnemius (p <0.05) Figure 3. Skeletal muscle protein synthesis is similar in Min mice at younger age. Wild type and ApcMin/+ mice were sacrificed at 10 weeks of age. Myofibrillar protein synthesis was measured in whole gastrocnemius. Values are ± SE, n = 3.

Figure 1.  

   

Figure 2.  

                   

Figure 3.