Protein quantification metabolic labeling

Figure 13.7 Schematic representation of quantification using metabolic labeling of relative abundances of proteins from two cell populations.

Several techniques have been developed for quantification using stable isotope dilution [57]. There are special considerations for the global addition of stable isotope labels before or after protein digestion, and the metabolic labeling of proteins in vivo, e.g., growing cells or even whole animals in which all proteins have been... 

Many analytical strategies have been developed for stable-isotope-based quantification of proteins, which all differ in the way stable isotopes are introduced into peptides or proteins (Fig. 15). They can be classified as (1) metabolic labeling, where cells acquire stable isotopes from the growth medium and incorporate them during protein biosynthesis , (2) enzymatic labeling, where stable isotopes are incorporated by an enzymatic reaction performed in vitro (3) chemical labeling, where stable isotopes are introduced by a chemical reaction in vitro and (4) spiking in a labeled reference compound. 

Intracellular fluxes can be estimated more precisely through 13C tracer experiments. Following 13C feeding to a cell it is possible to analyze metabolic products, such as amino acids, and measure 13C enriched patterns, so to be able to reconstruct the flux distribution from the measured data [91]. To obtain flux data from the labeling patterns, two techniques can be applied NMR [92, 93] and MS [94, 95]. Due to the low intracellular concentration of metabolites, these are often difficult to measure therefore the analysis of the labeling pattern of amino acids in proteins is used as input for flux quantification. Here proteins are hydrolyzed to release labeled amino acids and further analyzed by NMR of GC-MS. Once NMR or MS spectra are recorded, the next step is the quantitative interpretation of the isotopomer data by using mathematical models that describe the relationship between fluxes and the observed isotopomer abundance [96, 97], Some of the mathematical approaches used include cumulative isotopomer (cumomers) [98], bondomers [99], and fractional labeling [100], For a more comprehensive review on the methods we refer to Sauer [91]. 

The contribution of MS to identification of compounds and quantification of their concentration is complementary to other detection techniques and, despite being very practical and versatile, it remains fundamentally replaceable. However, knowledge of molecular weight is a prerequisite for techniques that rely on the synergies with stable isotopic tracers. In fact, powerful analytical methods exist to obtain important insights on cell dynamics from the ratiometric measurement of marked and not-marked species (or atoms). We cite, for example, (1) relative abundances of virtually all metabolites or proteins in two separate cultures are quantified based on the isotope dilution theory [43 5] (2) information on the mechanisms and kinetics of nonlinear chemical processes can be extracted from response tracer experiments [46 7] and (3) the labeling patterns in metabolic intermediates are used to resolve the relative rate in convergent reactions in vivo [48,49]. 

Techniques for quantitative ADME typically employ radiolabeled drugs to look at tissue distribution either in homogenized samples or by whole body autoradiography. Whole body autoradiography involving the location and quantification of radiolabeled material in thin frozen sections of tissue can be particularly useful. Plasma protein binding and metabolism studies also benefit from the use of radio-labeled material. 

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