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

  • Randall J. BatemanEmail author
  • Ling Y. Munsell
  • Xianghong Chen
  • David M. Holtzman
  • Kevin E. Yarasheski


In all biological systems, protein amount is a function of the rate of production and clearance. The speed of a response to a disturbance in protein homeostasis is determined by turnover rate. Quantifying alterations in protein synthesis and clearance rates is vital to understanding disease pathogenesis (e.g., aging, inflammation). No methods currently exist for quantifying production and clearance rates of low-abundance (femtomole) proteins in vivo. We describe a novel, mass spectrometry—based method for quantitating low-abundance protein synthesis and clearance rates in vitro and in vivo in animals and humans. The utility of this method is demonstrated with amyloid-β (Aβ), an important low-abundance protein involved in Alzheimer’s disease pathogenesis. We used in vivo stable isotope labeling, immunoprecipitation of Aβ from cerebrospinal fluid, and quantitative liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-tandem MS) to quantify human Aβ protein production and clearance rates. The method is sensitive and specific for stable isotope-labeled amino acid incorporation into CNS Aβ (+1% accuracy). This in vivo method can be used to identify pathophysiologic changes in protein metabolism and may serve as a biomarker for monitoring disease risk, progression, or response to novel therapeutic agents. The technique is adaptable to other macromolecules, such as carbohydrates or lipids.


Clearance Rate Fractional Synthesis Rate Fractional Clearance Rate Mass Isotopomer Distribution Analysis Human Neuroglioma Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Andersen, J. S.; Lam, Y. W.; Leung, A. K.; Ong, S. E.; Lyon, C. E.; Lamond, A. I.; Mann, M. Nucleolar Proteome Dynamics. Nature. 2005, 433, 77–83.CrossRefGoogle Scholar
  2. 2.
    Zhang, G.; Neubert, T. A. Automated Comparative Proteomics Based on Multiplex Tandem Mass Spectrometry and Stable Isotope Labeling. Mol. Cell. Proteomics. 2006, 5, 401–411.CrossRefGoogle Scholar
  3. 3.
    Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Mol. Cell. Proteomics. 2002, 1, 376–386.CrossRefGoogle Scholar
  4. 4.
    Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Matthews, D. E.; Yates, J. R., 3rd. Metabolic Labeling of Mammalian Organisms with Stable Isotopes for Quantitative Proteomic Analysis. Anal. Chem. 2004, 76, 4951–4959.CrossRefGoogle Scholar
  5. 5.
    Jaleel, A.; Nehra, V.; Persson, X. M.; Boirie, Y.; Bigelow, M.; Nair, K. S. In Vivo Measurement of Synthesis Rate of Multiple Plasma Proteins in Humans. Am. J. Physiol. Endocrinol. Metab. 2006, 291, E190-E197.CrossRefGoogle Scholar
  6. 6.
    Wolfe, R. R. Regulation of Skeletal Muscle Protein Metabolism in Catabolic States. Curr. Opin. Clin. Nutr. Metab. Care. 2005, 8, 61–65.CrossRefGoogle Scholar
  7. 7.
    Wolfe, R. R. Regulation of Muscle Protein by Amino Acids. J. Nutr. 2002, 132, 3219S-3224S.Google Scholar
  8. 8.
    San Pietro, A.; Rittenberg, D. A Study of the Rate of Protein Synthesis in Humans: II. Measurement of the Metabolic Pool and the Rate of Protein Synthesis. J. Biol. Chem. 1953, 201, 457–473.Google Scholar
  9. 9.
    Balagopal, P.; Rooyackers, O. E.; Adey, D. B.; Ades, P. A.; Nair, K. S. Effects of Aging on In Vivo Synthesis of Skeletal Muscle Myosin Heavy-chain and Sarcoplasmic Protein in Humans. Am. J. Physiol. Endocrinol. Metab. 1997, 273, E790-E800.Google Scholar
  10. 10.
    Yarasheski, K. E. Exercise, Aging, and Muscle Protein Metabolism. J. Gerontol. A Biol. Sci. Med. Sci. 2003, 58, M918-M922.CrossRefGoogle Scholar
  11. 11.
    Elias, N.; Patterson, B. W.; Schonfeld, G. In Vivo Metabolism of ApoB, ApoA-I, and VLDL Triglycerides in a Form of Hypobetalipoproteinemia Not Linked to the ApoB Gene. Arterioscler. Thromb. Vasc. Biol. 2000, 20, 1309–1315.CrossRefGoogle Scholar
  12. 12.
    Podlisny, M. B.; Lee, G.; Selkoe, D. J. Gene Dosage of the Amyloid Beta Precursor Protein in Alzheimer’s Disease. Science. 1987, 238, 669–671.CrossRefGoogle Scholar
  13. 13.
    Bateman, R. J.; Munsell, L. Y.; Morris, J. C.; Swarm, R.; Yarasheski, K. E.; Holtzman, D. M. Human Amyloid-beta Synthesis and Clearance Rates as Measured in Cerebrospinal Fluid In Vivo. Nat. Med. 2006, 12, 856–861.CrossRefGoogle Scholar
  14. 14.
    Murphy, M. P.; Uljon, S. N.; Fraser, P. E.; Fauq, A.; Lookingbill, H. A.; Findlay, K. A.; Smith, T. E.; Lewis, P. A.; McLendon, D. C.; Wang, R.; Golde, T. E. Presenilin 1 Regulates Pharmacologically Distinct Gamma-secretase Activities: Implications for the Role of Presenilin in Gamma-secretase Cleavage. J. Biol. Chem. 2000, 275, 26277–26284.CrossRefGoogle Scholar
  15. 15.
    DeMattos, R. B.; Bales, K. R.; Cummins, D. J.; Dodart, J. C.; Paul, S. M.; Holtzman, D. M. Peripheral Anti-A Beta Antibody Alters CNS and Plasma A Beta Clearance and Decreases Brain A Beta Burden in a Mouse Model of Alzheimer’s Disease. Proc. Natl. Acad. Sci. USA. 2001, 98, 8850–8855.CrossRefGoogle Scholar
  16. 16.
    Yarasheski, K. E.; Smith, S. R.; Powderly, W. G. Reducing Plasma HIV RNA Improves Muscle Amino Acid Metabolism. Am. J. Physiol. Endocrinol. Metab. 2005, 288, E278-E284.CrossRefGoogle Scholar
  17. 17.
    Smith, Q. R.; Momma, S.; Aoyagi, M.; Rapoport, S. I. Kinetics of Neutral Amino Acid Transport across the Blood—Brain Barrier. J. Neurochem. 1987, 49, 1651–1658.CrossRefGoogle Scholar
  18. 18.
    Krijgsveld, J.; Ketting, R. F.; Mahmoudi, T.; Johansen, J.; Artal-Sanz, M.; Verrijzer, C. P.; Plasterk, R. H.; Heck, A. J. Metabolic Labeling of C. elegans and D. melanogaster for Quantitative Proteomics. Nat. Biotechnol. 2003, 21, 927–931.CrossRefGoogle Scholar
  19. 19.
    Wang, R.; Sweeney, D.; Gandy, S. E.; Sisodia, S. S. The Profile of Soluble Amyloid Beta Protein in Cultured Cell Media: Detection and Quantification of Amyloid Beta Protein and Variants by Immunoprecipitation-Mass Spectrometry. J. Biol. Chem. 1996, 271, 31894–31902.CrossRefGoogle Scholar
  20. 20.
    Patterson, B. W. Use of Stable Isotopically Labeled Tracers for Studies of Metabolic Kinetics: An Overview. Metabolism. 1997, 46, 322–329.CrossRefGoogle Scholar
  21. 21.
    Yarasheski, K. E.; Smith, K.; Rennie, M. J.; Bier, D. M. Measurement of Muscle Protein Fractional Synthetic Rate by Capillary Gas Chromatography/Combustion Isotope Ratio Mass Spectrometry. Biol. Mass Spectrom. 1992, 21, 486–490.CrossRefGoogle Scholar
  22. 22.
    Hasten, D. L.; Morris, G. S.; Ramanadham, S.; Yarasheski, K. E. Isolation of Human Skeletal Muscle Myosin Heavy Chain and Actin for Measurement of Fractional Synthesis Rates. Am. J. Physiol. Endocrinol. Metab. 1998, 275, E1092-E1099.Google Scholar
  23. 23.
    Merchak, A.; Patterson, B. W.; Yarasheski, K. E.; Hamvas, A. Use of Stable Isotope Labeling Technique and Mass Isotopomer Distribution Analysis of [(13)C]Palmitate Isolated from Surfactant Disaturated Phospholipids to Study Surfactant In Vivo Kinetics in a Premature Infant. J. Mass Spectrom. 2000, 35, 734–738.CrossRefGoogle Scholar
  24. 24.
    Schulte, J. N.; Yarasheski, K. E. Effects of Resistance Training on the Rate of Muscle Protein Synthesis in Frail Elderly People. Int. J. Sport Nutr. Exerc. Metab. Suppl. 2001, 11, S111-S118.Google Scholar
  25. 25.
    Doherty, M. K.; Whitehead, C.; McCormack, H.; Gaskell, S. J.; Beynon, R. J. Proteome Dynamics in Complex Organisms: Using Stable Isotopes to Monitor Individual Protein Turnover Rates. Proteomics. 2005, 5, 522–533.CrossRefGoogle Scholar
  26. 26.
    Cargile, B. J.; Bundy, J. L.; Grunden, A. M.; Stephenson, J. L., Jr. Synthesis/Degradation Ratio Mass Spectrometry for Measuring Relative Dynamic Protein Turnover. Anal. Chem. 2004, 76, 86–97.CrossRefGoogle Scholar
  27. 27.
    Papageorgopoulos, C.; Caldwell, K.; Shackleton, C.; Schweingrubber, H.; Hellerstein, M. K. Measuring Protein Synthesis by Mass Isotopomer Distribution Analysis (MIDA). Anal. Biochem. 1999, 267, 1–16.CrossRefGoogle Scholar
  28. 28.
    Li, X. J.; Zhang, H.; Ranish, J. A.; Aebersold, R. Automated Statistical Analysis of Protein Abundance Ratios from Data Generated by Stable-Isotope Dilution and Tandem Mass Spectrometry. Anal. Chem. 2003, 75, 6648–6657.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  • Randall J. Bateman
    • 1
    • 4
    • 5
    Email author
  • Ling Y. Munsell
    • 2
  • Xianghong Chen
    • 2
  • David M. Holtzman
    • 1
    • 3
    • 4
    • 5
  • Kevin E. Yarasheski
    • 2
  1. 1.Department of NeurologyWashington University School of MedicineSt. Louis
  2. 2.Department of MedicineWashington University School of MedicineSt. LouisUSA
  3. 3.Department of Molecular Biology and PharmacologyWashington University School of MedicineSt. LouisUSA
  4. 4.Hope Center for Neurological DisordersWashington University School of MedicineSt. LouisUSA
  5. 5.Alzheimer’s Disease Research CenterWashington University School of MedicineSt. LouisUSA

Personalised recommendations