TY - JOUR
T1 - Stable Isotope Labeling Tandem Mass Spectrometry (SILT) to Quantify Protein Production and Clearance Rates
AU - Bateman, Randall J.
AU - Munsell, Ling Y.
AU - Chen, Xianghong
AU - Holtzman, David M.
AU - Yarasheski, Kevin E.
N1 - Funding Information:
This work was supported by grants from the American Academy of Neurology Foundation, National Institute on Aging Grant K08 AG-027091-01 and Alzheimer’s Disease Research Centers Grant P50 AG-05681, Blanchette Hooker Rockefeller Foundation, GCRC (MO1 RR00036), Mass Spectrometry Resource (RR000954), the Clinical Nutrition Research Unit (DK056341), and the Diabetes Research and Training Center (DK020579). The authors are grateful to the participant for his time, to Cambridge Isotope Laboratories (Ronald Trolard) for providing the 13 C 6 -phenylalanine, to Eli Lilly for providing m266 antibody, and to Dr. Robert Swarm for providing support for lumbar catheterization.
PY - 2007/6
Y1 - 2007/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=34248595908&partnerID=8YFLogxK
U2 - 10.1016/j.jasms.2007.02.009
DO - 10.1016/j.jasms.2007.02.009
M3 - Article
C2 - 17383190
AN - SCOPUS:34248595908
SN - 1044-0305
VL - 18
SP - 997
EP - 1006
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
IS - 6
ER -