Measuring protein synthesis using metabolic 2H labeling, high-resolution mass spectrometry, and an algorithm

Abstract

We recently developed a method for estimating protin dynamics in vivo with 2H2O using MALDI-TOF MS (Rachdaoui N. et al., MCP, 8, 2653-2662, 2009) and we confirmed that 2H-labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the 2H-enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In this study we have used nanospray LTQ-FTICR mass spectrometry to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor:product labeling ratio can be obtained by measuring the labeling of water and a protein(s) (or peptides) of interest, therein minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given 2H2O.

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

“…Because the plateau labeling (E ss ) depends on TBW labeling and the asymptotical number of exchanged H atoms (N) (13),…”

“…The number of exchanged hydrogen atoms in each analyzed peptide was calculated using software previously described (13). Briefly, this software generates the mass isotopomer distribution of a peptide based on plasma 2 H 2 O labeling and the number of incorporated 2 H atoms and compares that with experimentally measured plateau labeling of a peptide.…”

“…In addition, these techniques are not suited for human studies because they require the consumption of a fully 15 N-labeled diet. Recently we and others developed the 2 H 2 O metabolic labeling technique to measure protein turnover in free living animals (11)(12)(13)(14)(15). In rats that have been given intraperitoneal injection of 2 H 2 O, 2 H equilibrates with total body water and amino acids such as alanine within 10 and 20 min, respectively (16) (Fig.…”

“…Recently we demonstrated that the steady state 2 H labeling of most intracellular amino acids was achieved within 45 min of an intraperitoneal bolus of 2 H 2 O, suggesting that the transfer of amino acids to the polypeptide chain is the rate-limiting step in protein biosynthesis (12,17). The incorporation of multiple copies of Recently we developed a mathematical algorithm for the analysis of the mass isotopomer distribution and measurement of protein dynamics using high resolution FT-ICR MS (13). This approach is based on the concept that 2 H 2 O is the tracer precursor and the proteolytic peptide is the product, which accumulates multiple copies of 2 H from 2 H-labeled amino acids.…”

Metabolomics Research with Tandem Mass Spectrometry

“…In this section, we will briefly discuss the application of 13 C and 2 H isotope containing compounds to the quantification of metabolic fluxes through fatty acid oxidation and protein synthesis. First we consider 2 H 2 O-metabolic labeling based approach utilizing tandem mass spectrometry for protein turnover studies (Kasumov et al, 2011). Proteins exist in a dynamic state of equilibrium with their surrounding environment.…”