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

Abstract

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. Molecular & Cellular Proteomics 6:1416 -1427, 2007.As the field of proteomics matures as a discipline, there is an increasing realization of the importance of absolute as well as relative quantification, and considerable effort is being directed toward experimental strategies to achieve this goal. Most commonly, relative protein quantification by mass spectrometry has been based on differential stable isotope labeling implemented by metabolic incorporation (1, 2) or through derivatization strategies such as ICAT (3). The mass-coded abundance tagging method (4) avoids the use of stable isotopes but requires assumptions concerning mass spectrometric response factors. To achieve relative quantification of proteins without isotope labeling or chemical modification steps, quantitative comparisons have been made of equivalent sets of mass spectrometric data by considering peptide detectability in repetitively acquired spectra or by comparing integrated extracted ion chromatograms following liquid chromatography-mass spectrometry analyses (5).In principle, any of the approaches adopted for relative quantificatio...

Quantitative Analysis of Isotope Distributions In Proteomic Mass Spectrometry Using Least-Squares Fourier Transform Convolution

“…It is straightforward to define a variable fractional 13 Cenrichment in analogy to the fractional 15 N-labeling described above. The abundance vector for the new fractionally labeled C* atom is specified as: (33) These expressions provide an analytical description of a combination of fractional residue and fractional atom labeling that can be used for least-squares fitting of fractionally labeled peptides.…”

Protein quantification by selective isolation and fragmentation of isotopic pairs using FT-ICR MS

“…Chicken skeletal muscle protein and stable isotope [ 15 N]-and [ 13 C 6 ]-K/R-labeled chicken muscle QconCAT protein were prepared and quantified as described previously [6,7]. Labeled QconCAT was added in known amounts to unfractionated chicken muscle protein sample for quantitative analysis.…”

“…The producAddress reprint requests to Prof. Simon J. Gaskell, University of Manchester, School of Chemistry and Manchester Interdisciplinary Biocentre, John Garside Building, 131 Princess Street Manchester Ml 7DN, UK. E-mail: simon.gaskell@manchester.ac.uk tion of a QconCAT protein thereby enables absolute quantification of multiple proteins using their peptide surrogates encoded within the QconCAT [7]. In a typical QconCAT workflow those proteins for which absolute quantification is required are defined and one or more specific reference peptides (Q-peptides) are chosen for each protein [5].…”

“…Chemical synthesis and stable isotope incorporation of individual internal standards allow direct signal comparison, enabling the inference of protein quantities [4J. The production of a collection of internal standards in a single step has been enabled by the design of a DNA construct that is transcribed and translated into a protein concatamer, a technique referred to as QconCAT [5][6][7]. The produc-tion of a QconCAT protein thereby enables absolute quantification of multiple proteins using their peptide surrogates encoded within the QconCAT [7].…”

“…The production of a collection of internal standards in a single step has been enabled by the design of a DNA construct that is transcribed and translated into a protein concatamer, a technique referred to as QconCAT [5][6][7]. The produc-tion of a QconCAT protein thereby enables absolute quantification of multiple proteins using their peptide surrogates encoded within the QconCAT [7]. In a typical QconCAT workflow those proteins for which absolute quantification is required are defined and one or more specific reference peptides (Q-peptides) are chosen for each protein [5].…”