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

Abstract

Proteins serve many purposes by acting as structural supports, receptors, signaling molecules and enzymes, in addition, they facilitate nutrient transport and maintain immunological responses. Although the concentration of a given protein may not change appreciably over a short interval, proteins are continuously remodeled. In this chapter we consider how to study protein kinetics. Attention is directed towards two critical areas which include (i) the logic behind using different tracers and (ii) how to design and execute experiments that are compatible with proteome-based analyses. A practical illustration may highlight the importance of using isotope tracers to facilitate research in this area. For example, the concentration of circulating albumin provides a measure of protein nutritional status (and is a predictor of a patient’s recovery from disease), however, since the fractional turnover of albumin is relatively slow (~ 3 to 5% of the pool is newly made per day) several weeks of an intervention may be required to affect plasma levels. Recognizing that the concentration of albumin is a delayed-onset marker of nutritional status, investigators have used isotope tracers to determine the acute response of plasma albumin synthesis to a dietary manipulation, accordingly, one can make predictions regarding the efficacy of an intervention. Such studies rely on straightforward experimental designs. Namely, an investigator first decides on what amino acid will be used (e.g. 2H3leucine) and how will it be administered (e.g. a primed-constant infusion), samples are then collected for a given amount of time and a protein of interest (e.g. albumin) is isolated. Once isolated, the protein of interest is degraded (typically via acid hydrolysis) and the labeling of the free amino acid present in the plasma is compared to that of the amino acid that was bound in the protein, i.e. one determines the precursor:product labeling ratio. Although this scenario is relatively straightforward, our review considers the pros and cons surrounding the use of different tracers. In particular, we discuss recent advances in the use of stable