TY - JOUR

T1 - Determination of human muscle protein fractional synthesis rate

T2 - an evaluation of different mass spectrometry techniques and considerations for tracer choice

AU - Bornø, Andreas

AU - Hulston, Carl J

AU - van Hall, Gerrit

N1 - Copyright © 2014 John Wiley & Sons, Ltd.

PY - 2014/8

Y1 - 2014/8

N2 - In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically labeled amino acid will be incorporated within the few hours of a typical laboratory experiment. GC combustion isotope ratio MS (GC-C-IRMS) has thus far been considered the 'gold' standard for the precise measurements of these low enrichment levels. However, advances in liquid chromatography-tandem MS (LC-MS/MS) and GC-tandem MS (GC-MS/MS) have made these techniques an option for human muscle FSR measurements. Human muscle biopsies were freeze dried, cleaned, and hydrolyzed, and the amino acids derivatized using either N-acetyl-n-propyl, phenylisothiocyanate, or N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) for GC-C-IRMS, LC-MS/MS, and GC-MS/MS analysis, respectively. A second derivative, heptafluorobutyric acid (HFBA), was also used for GC-MS/MS analysis as an alternative for MTBSTFA. The machine reproducibility or the coefficients of variation for delta tracer-tracee-ratio measurements (delta tracer-tracee-ratio values around 0.0002) were 2.6%, 4.1%, and 10.9% for GC-C-IRMS, LC-MS/MS, and GC-MS/MS (MTBSTFA), respectively. FSR determined with LC-MS/MS compared well with GC-C-IRMS and so did the GC-MS/MS when using the HFBA derivative (linear fit Y = 1.08 ± 0.10, X + 0.0049 ± 0.0061, r = 0.89 ± 0.01, P < 0.0001). In conclusion, (1) IRMS still offers the most precise measurement of human muscle FSR, (2) LC-MS/MS comes quite close and is a good alternative when tissue quantities are too small for GC-C-IRMS, and (3) If GC-MS/MS is to be used, then the HFBA derivative should be used instead of MTBSTFA, which gave unacceptably high variability.

AB - In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically labeled amino acid will be incorporated within the few hours of a typical laboratory experiment. GC combustion isotope ratio MS (GC-C-IRMS) has thus far been considered the 'gold' standard for the precise measurements of these low enrichment levels. However, advances in liquid chromatography-tandem MS (LC-MS/MS) and GC-tandem MS (GC-MS/MS) have made these techniques an option for human muscle FSR measurements. Human muscle biopsies were freeze dried, cleaned, and hydrolyzed, and the amino acids derivatized using either N-acetyl-n-propyl, phenylisothiocyanate, or N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) for GC-C-IRMS, LC-MS/MS, and GC-MS/MS analysis, respectively. A second derivative, heptafluorobutyric acid (HFBA), was also used for GC-MS/MS analysis as an alternative for MTBSTFA. The machine reproducibility or the coefficients of variation for delta tracer-tracee-ratio measurements (delta tracer-tracee-ratio values around 0.0002) were 2.6%, 4.1%, and 10.9% for GC-C-IRMS, LC-MS/MS, and GC-MS/MS (MTBSTFA), respectively. FSR determined with LC-MS/MS compared well with GC-C-IRMS and so did the GC-MS/MS when using the HFBA derivative (linear fit Y = 1.08 ± 0.10, X + 0.0049 ± 0.0061, r = 0.89 ± 0.01, P < 0.0001). In conclusion, (1) IRMS still offers the most precise measurement of human muscle FSR, (2) LC-MS/MS comes quite close and is a good alternative when tissue quantities are too small for GC-C-IRMS, and (3) If GC-MS/MS is to be used, then the HFBA derivative should be used instead of MTBSTFA, which gave unacceptably high variability.

U2 - 10.1002/jms.3387

DO - 10.1002/jms.3387

M3 - Journal article

C2 - 25044894

VL - 49

SP - 674

EP - 680

JO - Journal of Mass Spectrometry

JF - Journal of Mass Spectrometry

SN - 1076-5174

IS - 8

ER -