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

Abstract

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.

Integrative structure and functional anatomy of a nuclear pore complex

“…Mass spectrometry quantification of the relative amounts of each nucleoprotein in the purified NPC complex was performed using two specifically designed, heavy-labeled synthetic internal standards or QconCATs 51,54 (Extended Data Fig. 2D–E) formed by concatenated quantotypic nucleoporin peptides.…”

“…The E. coli codon optimized sequences were cloned into: i) plasmid pET15-b (as a NcoI-XhoI fragment) in the case of QconCAT-A; and ii) pGEX6p-1 (as a BamHI-XhoI fragment) in the case of QconCAT-B, resulting in the expression of a 68.1 kDa protein with a n-terminal GST tag that was mainly used as a sacrificial peptide 56 . The QconCAT proteins were expressed by growing 300 mL of BL21 E. coli cells at 37°C to OD 600 = 0.6 in minimal M9 media without ammonium chloride 51,54 supplemented with light amino acids and 0.5 mg/mL of heavy arginine and lysine (L-arginine:HCl 13C6; L-lysine:2HCl 13C6, Cambridge Isotope Laboratories Inc.). IPTG (1 mM) was used to induce expression of the constructs for 3 hours at 37°C.…”

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.…”

“…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 DNA coding sequences for these peptides are concatenated, inserted into a vector, and expressed in Escherichia coli to form an artificial QconCAT protein. Growth of the transfected E. coli in stable isotope-containing media yields labeled QconCAT protein [5,6]. To achieve absolute quantification, the QconCAT protein is introduced, in a known amount, into the biological sample of interest and proteins are digested with trypsin to yield Qpeptides alongside native peptides.…”