Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates

Abstract

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have developed a simple method to isolate myosin heavy chain (MHC) and actin from small (60-80 mg) human skeletal muscle samples for the determination of their fractional synthesis rates. The amounts of MHC and actin isolated are adequate for the quantification of [ 13 C]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Fractional synthesis rates of mixed muscle protein (MMP), MHC, and actin were determined in six healthy young subjects (27 ± 1 yr) after they received a 14-h intravenous infusion (prime = 7.58 μmol/kg body wt, constant infusion = 7.58 μmol·kg body wt −1 ·h −1 ) of [1-13 C]leucine. The fractional synthesis rates of MMP, MHC, and actin were found to be 0.0468 ± 0.0048, 0.0376 ± 0.0033, and 0.0754 ± 0.0078%/h, respectively. Overall, the synthesis rate of MHC was 20% lower (P = 0.012), and the synthesis rate of actin was 61% higher (P = 0.060, not significant) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC-C-IRMS provides important information about the synthesis rates of these specific contractile proteins, as opposed to the more general information provided by the determination of MMP synthesis rates.Keywords muscle protein synthesis; amino acid metabolism; protein metabolism; stable isotope tracers; mass spectrometry MOST STUDIES that have used stable isotope tracer methodology to determine the fractional synthesis rate of human muscle protein have reported the rate of "mixed" muscle protein (MMP) synthesis (14,15,23,24,26,27). A problem with the measurement of MMP synthesis is that it reflects the average rate of synthesis of several muscle proteins in the sample (i.e., contractile, enzymatic, mitochondrial). Changes in the synthesis rate of Copyright © 1998 the American Physiological Society Address for reprint requests: K. E. Yarasheski, Washington Univ. School of Medicine, 660 S. Euclid Ave., Box 8127, St. Louis, MO 63110.. NIH Public Access Author ManuscriptAm J Physiol. Author manuscript; available in PMC 2014 August 21.Published in final edited form as: Am J Physiol. 1998 December ; 275(6 0 1): E1092-E1099. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript individual contractile proteins could be confounded by corresponding or opposing changes in the synthesis rates of other proteins in the MMP sample. In particular, Rooyackers et al. (19) found that the rate of mitochondrial protein synthesis was ~95% higher than that of MMP in young subjects. Therefore, isolating specific contractile proteins from human muscle samples obtained during a stable isotopically labeled amino acid (e.g., [1-13 C]leucine) administration protocol, and measuring the amount of labeled amino acid incorporated into these specific contractile proteins by use of gas isotope ratio mass spectrometry (IRMS) would provide a more refined approach to in vivo studies of muscle contractile protein metabolism.Recent effort...

Stable isotope labeling tandem mass spectrometry (SILT) to quantify protein production and clearance rates

“…The calculated A␤ FSRs were similar, accounting for the twofold higher 13 C 6 -phenylalanine enrichment in the CSF precursor during the infusion and the twofold greater slope of the 13 C 6 -phenylalanine incorporation into A␤ [17][18][19][20][21][22][23][24][25][26][27][28] versus time curves compared to leucine. The A␤ FSR was 4.8%/h as measured by 13 C 6 -leucine-labeled A␤ and 5.8%/h from 13 C 6 -phenylalanine-labeled°A␤°(Figure°5).°The°A␤°FCR was 3.9%/h as measured by 13 C 6 -leucine-labeled A␤ and could not be measured from 13 C 6 -phenylalaninelabeled A␤ because of only 36 h of sampling.…”

“…Labeled cultured media was serially diluted with unlabeled media to generate samples for a standard curve. A␤ was immunoprecipitated from the media, trypsin digested, and A␤ [17][18][19][20][21][22][23][24][25][26][27][28] fragments were analyzed on a LCQ-ESI-MS and the tandem mass spectra ions were quantitated using custom-written software. The predicted percentage labeled A␤ versus the measured value is shown with a linear regression line.…”

“…and the presence of these naturally occurring isotopes, A␤ was enzymatically cleaved into fragments (A␤ 1-5 , A␤ 6 -16 , A␤ [17][18][19][20][21][22][23][24][25][26][27][28] , and A␤ 29 -40/42 ) using trypsin, before mass spectrometry analysis. The A␤ [17][18][19][20][21][22][23][24][25][26][27][28] fragment has a nominal mass of 1324.6 Daltons (D), contains one leucine residue, and was detected using MALDI-TOF-MS, as a singly charged species at m/z 1325.6 [MϩH] ϩ1 . The A␤ 6 -16 [17][18][19][20][21][22][23][24][25][26][27][28] peptide, and that this high level of 13 C 6 -leucine incorporation could be easily detected using MALDI-TOF-MS.…”

“…After the isolation and detection of labeled and unlabeled A␤, the amount of labeled and unlabeled A␤ [17][18][19][20][21][22][23][24][25][26][27][28] peptides was quantified using LC-ESI-tandem MS with quantitation of the full-scan tandem MS ions. The product ions detected by tandem mass spectrometry specifically identified the amino acid sequence for the labeled peptides based on the signature mass plus 6 D. This provided a highly specific "fingerprint" of the A␤ [17][18][19][20][21][22][23][24][25][26][27][28] in both labeled and unlabeled forms.…”

“…To assess the ratio of labeled to unlabeled A␤, the signal intensities from the product ions obtained in the tandem MS experiments were used to quantify the ratio of labeled to unlabeled A␤ [17][18][19][20][21][22][23][24][25][26][27][28] . The tandem MS spectra were averaged over the time that A␤ eluted from the LC column (number of tandem MS scans ϭ 5) for both labeled and unlabeled tandem MS spectra.…”