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

Abstract

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.

Integrative structure and functional anatomy of a nuclear pore complex

“…2A): (1) use of synthetic concatemers of tryptic peptides or QconCATs 51 to define the relative stoichiometry of each component by quantitative MS in affinity-captured NPCs; (2) In vivo calibrated imaging analysis of GFP-tagged Nups 52 , to quantify the absolute copy number per NPC of Nups selected to represent each major module of the NPC; and (3) Charge Detection MS to measure the total mass of affinity-captured NPCs 53 . For the calculation of the integrative NPC structure, the final copy numbers were rounded to fit the known NPC C 8 -symmetry and these values are indicated in Supplementary Table 2A.…”

“…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

“…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.…”

“…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].…”