Proteomics Moves From Expression to Turnover

Abstract and Introduction

Abstract

Proteomics is a rapidly developing discipline that seeks to understand the role of proteins in the wider biological context. In order to take a holistic view of a biological system, it is vital that we can elucidate the dynamics of the proteome. In this article, we have outlined the recent advances in experimental strategies for measuring protein synthesis and degradation on a proteome-wide scale. The application of mass spectrometry and non-mass spectrometric-based approaches in this field of research has been discussed. The article also explores the challenges associated with these types of analyses and the development of appropriate bioinformatic resources for interrogating the complex datasets that are generated.

Introduction

Proteomics is a key technology used to probe the biology of living systems. It has developed rapidly from the simple identification of proteins to the high-throughput characterization and quantification of large cohorts of proteins.^[1,2] Proteomics seeks to provide a functional link between expressed genes and phenotypic outcomes. Unlike the genome, which is considered the blueprint of an organism, the proteome is not a predetermined, static entity. The proteome is in constant flux, which is influenced by environmental conditions, cellular function, developmental status and extracellular challenge. In addition to changes in protein concentration, the function of a protein can also be modulated by post-translation modifications such as phosphorylation, ubiquitination, glycosylation and proteolysis. Even when a cell is in a position of apparent steady state, the protein complement is constantly changing, with new proteins being synthesized and older proteins being degraded and recycled to minimize damage to the cell.^[3]

Moving from a single 'snapshot' of the proteome to understanding the dynamic processes requires the use of sophisticated analytical and bioinformatic resources. Mass spectrometry (MS) is a key technology in the field of proteomics, with recent developments in instrumentation allowing the proteome to be probed to unprecedented levels. These advances, coupled with the ability to obtain accurate quantitative data for large cohorts of proteins,^[4,5] have led to an explosion in proteomic applications. In this article, we will focus on recent developments in the methods and technologies used to explore the dynamics of cellular protein turnover in biological systems.

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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