Proteomics Moves From Expression to Turnover

Defining Protein Turnover

Protein turnover refers to the continual renewal or replacement of protein. It is defined by the balance between protein synthesis and protein degradation.^[6] During periods of steady state, the overall rate of protein synthesis is equal to the rate of protein degradation. When an organism or cell is growing, the rate of protein synthesis exceeds that of protein degradation (anabolism), while the opposite is true during periods of catabolism. This balancing act allows the cell to regulate function and provides flexibility to rapidly respond to cellular events. Protein synthesis is driven by the transcriptome and, although the concentration of mRNA transcript influences the downstream concentration of cognate protein, there is not always a direct correlation.^[7–9] The rate of protein synthesis is also influenced by initiation of transcription and ribosomal activity. The ability to accurately define the rate of protein synthesis has wide applications in our understanding of genotype–phenotype relationships and in the study of pathophysiological processes. Proteins can be degraded by a number of pathways in eukaryotes. The ubiquitin proteasome system and autophagy have been of primary interest in recent years. The ubiquitin proteasome system allows for the selective degradation of proteins that have been tagged by a polyubiquitin chain and directed to the 26S proteasome. Autophagy is believed to be a system of bulk protein, and indeed organelle, degradation that is triggered during times of stress, such as starvation. The ability for a protein to replenish its protein population is essential for maintaining health and viability. Dysfunction of protein turnover has been linked to a range of diseases, particularly those prominent in an aging population, including Parkinson's disease,^[10,11] Alzheimer's disease^[12,13] and cancer.^[14,15]

Measuring protein turnover is complex. Even in simple unicellular organisms such as bacteria and yeast, there are many up- and down-stream processes that must be considered. The major premise of any approach to calculate protein synthesis or degradation rates is the measurement of incorporation or loss of a labeled precursor into the protein. In general, it is imperative that we are able to accurately determine the extent to which the precursor pool has been labeled. Although the true precursor pool for protein synthesis is the aminoacyl-tRNA pool,^[16–18] these are in low abundance in cells and are difficult to quantify with precision.^[19–21] Therefore, it is common to see amino acids used as a surrogate for the aminoacyl-tRNA pool.

When using cells in culture, it is relatively easy to control the specific (radio)activity (SA) or relative isotope abundance (RIA) of the precursor by supplementation of the external medium with a substantial excess of the radiolabeled or stable isotope-labeled compound. The SA/RIA quickly reaches a plateau to unity, and subsequent sampling of the labeled proteins then permits assessment of the rate of turnover of the proteins. However, in more complex multicellular species, interactions occur between the different cellular pools. This leads to an inability to precisely control the precursor RIA or SA to the same extent, and therefore requires the labeling of the precursor pool to be determined experimentally (Figure 1).

(Enlarge Image)

Figure 1.

The requirement for relative isotope abundance calculation in multicellular systems. Proteins are continually being synthesized and degraded by cells. In animal systems, unlabeled amino acids are provided from the recycling of proteins from other tissues. When using stable isotope labeling approaches, it is therefore critical that the RIA of the precursor is accurately defined. Stable isotope-labeled amino acids are introduced to the system at a defined RIA. In this example, dietary RIA = 0.5. Dilution from other tissues will lower the true precursor RIA for protein synthesis. The precursor RIA can be calculated from the relative intensity of the mass spectral ions generated from the proteins of interest.
RIA: Relative isotope abundance.

Prior to the development of proteomic technologies, the principal way of determining protein turnover in complex organisms, particularly in human subjects, was to introduce tracers in either the flooding dose method or by continuous infusion. The tracer, usually a radioisotope, is introduced to the subject either via a bolus injection or through an intravenous line. Proteins can then be separated by 1 or 2DE and the level of incorporation of the tracer is then measured directly from the tissues of interest, typically by autoradiography. The practical aspects, advantages and disadvantages of these methods have been described extensively elsewhere.^[3,22] The primary limitation of these approaches is that quantitative information is only easily obtained for bulk protein turnover. The lack of protein-specific information arose from the historical difficulties of labeling proteins to a sufficient degree that individual protein turnover could be assessed. Moreover, global studies were hindered by the ability to establish the true specific activity of the precursor pool and a lack of methods to measure the kinetics of long-lived proteins. Synthesis and degradation rates of specific individual proteins have been previously determined, but the approaches used do not lend themselves to the analysis of large cohorts of proteins simultaneously. Further issues relate to the observation that amino acids may, in some cases, influence protein turnover in intact organisms. Buse and Reid reported the potential for leucine to regulate protein turnover in muscle,^[23] and Pannemans et al. reported that certain tracers can affect protein turnover in elderly women.^[24]

Cite this: Proteomics Moves From Expression to Turnover - Medscape - Jun 01, 2011.

Table 1. Summary of approaches used to determine protein turnover.
Method	Application	Label	System	Ref.
Dynamic SILAC	Determination of protein degradation rates by MS	Amino acid	Yeast Chicken Human cell line	[31,34,42]
PC/QMS	Pulse/chase approach to determine ribosome assembly dynamics	Amino acid	Escherichia coli	[45]
Combined SILAC/iTRAQ	Protein dynamics under non-steady-state conditions	Amino acid	Streptomyces coelicor	[46]
Pulsed SILAC	Measures the relative rate of protein synthesis under different experimental conditions	Amino acid	HeLa cells Yeast	[43]
Ratio methods	Profiling protein turnover under different experimental conditions	Ammonium sulfate	Mycobacterium smegmatis	[47]
SINEW	Whole animal protein turnover	Water	Mouse	[48]
Plant dynamics	Measuring proteome turnover in plants	Water	Arabidopsis	[50]
TAP tagging	Non-mass spectrometric method to profile protein stability		Yeast	[51]
Global Protein Stability	Fluorescent assay using microarrays		Human	[53]

iTRAQ: Isobaric tag for relative and absolute quantitation; MS: Mass spectrometry; PC/QMS: Pulse-chase MS approach; SILAC: Stable isotope labeling of amino acids in cell culture; SINEW: Stable isotope labeling of nonessential amino acids with heavy water; TAP: Tandem affinity purification.

Table 2. Quantification software programs for mass spectrometry.
Name of program	Application	Availability	Ref.
MASCOT	Integral to the MASCOT search engine Generic to all instrument vendors Licensed software	Commercial	[101]
MaxQuant	Primarily for Thermo FT and Orbitrap instruments. Has a combined search and quantification package, which allows the user to upload protein databases. Incorporates GO analysis	Freeware	[102]
MSQuant	Suitable for Applied Biosystems, Thermo and Waters instruments. Supports amino acid and 15N labeling	Freeware	[103]
RELEX	Specific to Thermo instruments	Freeware	[104]
XPRESS	Available as part of the TransProteome Pipeline package. Generic to all instrument vendors	Freeware	[105]

GO: Gene ontology.

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