Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes.

An important area of proteomics involves the need for quantification, whether relative or absolute. Many methods now exist for relative quantification, but to support biomarker proteomics and systems biology, absolute quantification rather than relative quantification is required. Absolute quantification usually involves the concomitant mass spectrometric determination of signature proteotypic peptides and stable isotope-labeled analogs. However, the availability of standard labeled signature peptides in accurately known amounts is a limitation to the widespread adoption of this approach. We describe the design and synthesis of artificial QconCAT proteins that are concatamers of tryptic peptides for several proteins. This protocol details the methods for the design, expression, labeling, purification, characterization and use of the QconCATs in the absolute quantification of complex protein mixtures. The total time required to complete this protocol (from the receipt of the QconCAT expression plasmid to the absolute quantification of the set of proteins encoded by the QconCAT protein in an analyte sample) is approximately 29 d.

Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides.

Absolute quantification in proteomics usually involves simultaneous determination of representative proteolytic peptides and stable isotope-labeled analogs. The principal limitation to widespread implementation of this approach is the availability of standard signature peptides in accurately known amounts. We report the successful design and construction of an artificial gene encoding a concatenation of tryptic peptides (QCAT protein) from several chick (Gallus gallus) skeletal muscle proteins and features for quantification and purification.

Metabolic labeling of proteins for proteomics.

Realization of the advantages of stable isotope labeling for proteomics has emerged gradually. However, many stable isotope label approaches rely on labeling in vitro using complex and sometimes expensive reagents. This review discusses strategies for labeling protein in vivo through metabolic incorporation of label into protein. This approach has many advantages, is particularly suited to single cells grown in culture (prokaryotic or eukaryotic), but is nonetheless subject to a number of complicating factors that must be controlled so that meaningful experiments can be conducted. Confounding issues include the metabolic lability of the amino acid precursor, incomplete labeling, and the role of protein turnover in labeling kinetics. All of these are controllable, provided that appropriate precautions are adopted.

The proteome of chicken skeletal muscle: changes in soluble protein expression during growth in a layer strain.

The whole animal, and the pectoralis muscle in particular, grows at a greatly enhanced rate in chickens selected for meat production (broilers) when compared to those selected for egg production (layers). As part of an ongoing study to analyse muscle protein dynamics under conditions of rapid growth, we have embarked upon a preliminary characterisation of the proteome of layer chicken pectoralis muscle, at specified time-points from 1 to 27 days after hatching. Soluble extracts of muscle homogenates were separated by two-dimensional (2-D) gel electrophoresis and selected spots were analysed by in-gel tryptic digestion and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Of 90 spots, 51 gave mass spectra that matched to existing chicken proteins present in on-line databases, 12 matched equivalent proteins from non-avian species and 11 yielded good quality spectra but were unable to be matched against existing databases. For many of these proteins, growth over 27 days elicited dramatic changes in relative expression levels. Chicken skeletal muscle offers an excellent system for developmental proteomics.

Dynamics of protein turnover, a missing dimension in proteomics.

Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucose-limited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species.

Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting.

Peptide mass fingerprinting (PMF) is a powerful technique for identification of proteins derived from in-gel digests by virtue of their matrix-assisted laser desorption/ionization-time of flight mass spectra. However, there are circumstances where the under-representation of peptides in the mass spectrum and the complexity of the source proteome mean that PMF is inadequate as an identification tool. In this paper, we show that identification is substantially enhanced by inclusion of composition data for a single amino acid. Labelling in vivo with a stable isotope labelled amino acid (in this paper, decadeuterated leucine) identifies the number of such amino acids in each digest fragment, and show a considerable gain in the ability of PMF to identify the parent protein. The method is tolerant to the extent of labelling, and as such, may be applicable to a range of single cell systems.