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

Abstract

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-␤ (A␤), an important low-abundance protein involved in Alzheimer's disease pathogenesis. We used in vivo stable isotope labeling, immunoprecipitation of A␤ from cerebrospinal fluid, and quantitative liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-tandem MS) to quantify human A␤ protein production and clearance rates. The method is sensitive and specific for stable isotope-labeled amino acid incorporation into CNS A␤ (Ϯ1% 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. (J Am Soc Mass Spectrom 2007, 18, 997-1006

Proteome Kinetics: Coupling the Administration of Stable Isotopes with Mass Spectrometry-Based Analyses

“…the labeled molecules divided by the sum of labeled and unlabeled molecules) (Dwyer et al 2002;Lichtenstein et al 1990;Magkos, Patterson, and Mittendorfer 2007). We agree that in cases where one infuses a labeled amino acid for a given time and then stops the infusion of the tracer that there will be a decrease in the labeling of a given protein over time ( Figure 3) (Bateman et al 2006;Bateman et al 2007). However, assuming that protein breakdown is a random process, i.e.…”

“…As noted above, the incorporation of a tracer into a protein of interest can be used to estimate the rate of synthesis, can one estimate the rate of protein breakdown by measuring the elimination of a tracer from a protein of interest? We believe that the answer is "no", or at the very least it is not as straightforward as reports in the literature (Bateman et al 2006;Bateman et al 2007). Readers should consider how measurements of isotopic labeling are typically performed and how data are expressed.…”

Protein production is an early biomarker for RNA‐targeted therapies

“…Because mRNA translation leads to the production of new protein, protein‐based biomarkers in CSF represent an attractive avenue to pursue target engagement for RNA‐targeted therapeutics. In this study, we used nonradioactive, stable isotope labeling with an essential amino acid 17, 18 to identify inhibition of new protein production after ASO treatment. Using two preclinical models of ASO targets in clinical trials for neurodegenerative disease, we tested the hypotheses that stable isotope labeling with 13 C 6 ‐Leucine will: (1) identify pharmacodynamics at an earlier time point than protein concentration‐based measures and (2) show measurable effects in CSF to support translation of this assay approach from animal models to human CNS.…”

In vivo kinetic approach reveals slow SOD1 turnover in the CNS

“…However, measuring turnover rates of proteins in an immortalized, rapidly dividing cell culture model has limitations and shows discrepancies when turnover rates of identical proteins are compared between tissues and cell culture models (4). The development of stable isotope-labeling kinetics (SILK) has enabled the study of protein turnover rates in vivo using a safe, stable isotope amino acid tracer that is incorporated into newly synthesized proteins and can be quantitatively measured by mass spectrometry -a technique that has been highly successful in studies of amyloid-β in human cerebral spinal fluid (CSF) and in brains from animal models (5)(6)(7)(8)(9)(10)(11)(12). In these studies, a relatively short (9-hour) intravenous infusion of the stable isotope 13 C 6 -leucine resulted in adequate labeling of the rapidly turned over amyloid-β (half-life of 8 hours).…”

“…The calculation of protein turnover by administering stable isotope-labeled amino acids or D 2 O over time has been demonstrated in numerous cell culture models and in human CSF and plasma proteins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)23). Stable isotope-labeled amino acids are biologically identical to their naturally occurring counterparts and, unlike radiolabeled amino acids, are innocuous to both the system being studied and the experimental environment.…”