Deborah M. Simpson

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 ∼29 d.

Absolute Multiplexed Quantitative Analysis of Protein Expression during Muscle Development Using QconCAT

Stable isotope-labeled proteotypic peptides are used as surrogate standards for absolute quantification of proteins in proteomics. However, a stable isotope-labeled peptide has to be synthesized, at relatively high cost, for each protein to be quantified. To multiplex protein quantification, we developed a method in which gene design de novo is used to create and express artificial proteins (QconCATs) comprising a concatenation of proteotypic peptides. This permits absolute quantification of multiple proteins in a single experiment. This complete study was constructed to define the nature, sources of error, and statistical behavior of a QconCAT analysis. The QconCAT protein was designed to contain one tryptic peptide from 20 proteins present in the soluble fraction of chicken skeletal muscle. Optimized DNA sequences encoding these peptides were concatenated and inserted into a vector for high level expression in Escherichia coli. The protein was expressed in a minimal medium containing amino acids selectively labeled with stable isotopes, creating an equimolar series of uniformly labeled proteotypic peptides. The labeled QconCAT protein, purified by affinity chromatography and quantified, was added to a homogenized muscle preparation in a known amount prior to proteolytic digestion with trypsin. As anticipated, the QconCAT was completely digested at a rate far higher than the analyte proteins, confirming the applicability of such artificial proteins for multiplexed quantification. The nature of the technical variance was assessed and compared with the biological variance in a complete study. Alternative ionization and mass spectrometric approaches were investigated, particularly LC-ESI-TOF MS and MALDI-TOF MS, for analysis of proteins and tryptic peptides. QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.

Abundance of tegument surface proteins in the human blood fluke Schistosoma mansoni determined by QconCAT proteomics

The schistosome tegument provides a major interface with the host blood stream in which it resides. Our recent proteomic studies have identified a range of proteins present in the complex tegument structure, and two models of protective immunity have implicated surface proteins as mediating antigens. We have used the QconCAT technique to evaluate the relative and absolute amounts of tegument proteins identified previously. A concatamer comprising R- or K-terminated peptides was generated with [(13)C(6)] lysine/arginine amino acids. Two tegument surface preparations were each spiked with the purified SmQconCAT as a standard, trypsin digested, and subjected to MALDI ToF-MS. The absolute amounts of protein in the biological samples were determined by comparing the areas under the pairs of peaks, separated by 6m/z units, representing the light and heavy peptides derived from the biological sample and SmQconCAT, respectively. We report that aquaporin is the most abundant transmembrane protein, followed by two phosphohydrolases. Tetraspanin Tsp-2 and Annexin-2 are also abundant but transporters are scarce. Sm200 surface protein comprised the bulk of the GPI-anchored fraction and likely resides in the secreted membranocalyx. Two host IgGs were identified but in amounts much lower than their targets. The findings are interpreted in relation to the models of protective immunity.

Absolute Quantification of the Glycolytic Pathway in Yeast: DEPLOYMENT OF A COMPLETE QconCAT APPROACH

The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.

QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification.

AbstractSystems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway. FigureWorkflow for QconCAT mediated protein quatification

QCAL-a Novel Standard for Assessing Instrument Conditions for Proteome Analysis

If proteome datasets are to be collated, shared, and merged for higher level proteome analyses, there is a need for generally accepted strategies and reagents for optimization and standardization of instrument performance. At present, there is no single protein or peptide standard set that is capable of assessing instrument performance for peptide separation and analysis in this manner. To create such a standard, we have used the recently described QconCAT methodology to generate an artificial protein, QCAL. This protein, a concatenation of tryptic peptides that is expressed in E. coli, provides a stoichiometrically controlled mixture of peptides that are amenable to analysis by all commonly used instrumentation platforms for proteomics.

Formation of 3-nitrotyrosines in carbonic anhydrase III is a sensitive marker of oxidative stress in skeletal muscle

Oxidation of skeletal muscle proteins has been reported to occur following contractions, with ageing, and with a variety of disease states, but the nature of the oxidised proteins has not been identified. A proteomics approach was utilised to identify major proteins that contain carbonyls and/or 3‐nitrotyrosine (3‐NT) groups in the gastrocnemius (GTN) muscles of adult (5–11 months of age) and old (26–28 months of age) wild type (WT) mice and adult mice lacking copper, zinc superoxide dismutase (Sod1−/− mice), manganese superoxide dismutase (Sod2+/− mice) or glutathione peroxidase 1 (GPx1−/− mice). In quiescent GTN muscles of adult and old WT mice, protein carbonylation and/or formation of 3‐NT occurred in several proteins involved in glycolysis, as well as creatine kinase and carbonic anhydrase III. Following contractions, the 3‐NT intensity was increased in specific protein bands from GTN muscles of both adult and old WT mice. In quiescent GTN muscles from adult Sod1−/−, Sod2+/− or GPx1−/− mice compared with age‐matched WT mice only carbonic anhydrase III showed a greater 3‐NT content. We conclude that formation of 3‐NT occurs readily in response to oxidative stress in carbonic anhydrase III and this may provide a sensitive measure of oxidative damage to muscle proteins.

Acetone Precipitation of Proteins and the Modification of Peptides

Acetone precipitation is a common method for precipitation and concentration of proteins. We show here that a trace amount of residual acetone in the precipitated protein, can, after proteolysis, lead to selective modification of peptides predominantly those in which a glycine residue is the second amino acid, probably generating a relatively stable derivative that, under gas phase conditions, generates a y(1) ion of the same mass as proline. This modification is detectable by either MALDI-ToF or ESI-ion trap mass spectrometry and under normal sample preparation conditions is incomplete. The derivatization occurs in the condensed phase and is sufficiently stable that the modified peptide can elute on reversed phase chromatography at a different time to the unmodified peptide. Acetone precipitation is such a commonly used procedure in protein sample preparation for proteomics that some caution may be warranted. A significant number of peptides (about 5% of a typical proteome) meet the requirements for this reaction and could, therefore, change the outcome of studies.

Urinary Lipocalins in Rodenta:is there a Generic Model?

It is increasingly clear that mediation of chemical signals is not the exclusive domain of low molecular volatile or water soluble metabolites. Pheromone binding proteins play an important role in mediating the activity of low molecular weight compounds, while proteins and peptides can also act as information molecules in their own right. Understanding of the role played by proteins in scents has been derived largely from the study of Major Urinary Proteins (MUPs) in the mouse (Mus musculus domesticus) and the rat (Rattus norvegicus). As part of an ongoing programme to explore the diversity and complexity of urinary proteins in rodents, we have applied a proteomics-based approach to the analysis of urinary proteins from a wider range of rodents. These data suggest that many species express proteins in their urine that are structurally similar to the MUPs, although there is considerable diversity in concentration, in sexual dimorphism and in polymorphic complexity. This is likely to reflect a high degree of species-specificity in communication and the information that these proteins provide in scent signals.

Anatomical heterogeneity of tendon: Fascicular and interfascicular tendon compartments have distinct proteomic composition

Tendon is a simple aligned fibre composite, consisting of collagen-rich fascicles surrounded by a softer interfascicular matrix (IFM). The composition and interactions between these material phases are fundamental in ensuring tissue mechanics meet functional requirements. However the IFM is poorly defined, therefore tendon structure-function relationships are incompletely understood. We hypothesised that the IFM has a more complex proteome, with faster turnover than the fascicular matrix (FM). Using laser-capture microdissection and mass spectrometry, we demonstrate that the IFM contains more proteins, and that many proteins show differential abundance between matrix phases. The IFM contained more protein fragments (neopeptides), indicating greater matrix degradation in this compartment, which may act to maintain healthy tendon structure. Protein abundance did not alter with ageing, but neopeptide numbers decreased in the aged IFM, indicating decreased turnover which may contribute to age-related tendon injury. These data provide important insights into how differences in tendon composition and turnover contribute to tendon structure-function relationships and the effects of ageing.