We report a new approach to the assembly and construction of QconCATs, quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. The new approach is based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. It offers a major gain in flexibility of QconCAT implementation and enables rapid and efficient editability that permits, for example, substitution of one peptide for another. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short also allows a second cycle of assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards.

The efficiency of stable-isotope incorporation of ALACAT synthesized in E. coli cell-free system was determined using LC-SRM analysis. Prior to SRM analysis, ALACAT protein B1 separated by SDS-PAGE was digested with trypsin in a gel and purified with a STAGE tip containing an Empore SDB-XC disc as described previously. The purified peptide sample was dissolved in 20 µL of 0.1% TFA solution, of which 1 µL was injected into an Eksigent nanoLC system coupled to a SCIEX QTRAP 5500 mass spectrometer. For LC separation, solvent A was 0.1% (v/v) formic acid, and solvent B was 0.1 % (v/v) formic acid/80 % (v/v) acetonitrile. The sample was desalted using a 200 µm i.d. × 0.5 mm cHiPLC trap column (SCIEX) at a flow rate of 5 μL/minute for 10 minutes using 0.1 % (v/v) TFA and then transported to a fused-silica capillary column packed with C18 resin (12.5 cm × 75 µm i.d.; Nikkyo Technos) at a flow rate of 300 nL/minute according to the following gradient schedule: 0–15 minutes, 2–50 % B; 15–18 minutes, 50–90 % B; hold at 90 % B for 6 minutes; and re-equilibrate at 2% B for 20 minutes. SRM analysis was conducted in positive ion mode with the following parameters: ion spray voltage = 2300 V; curtain gas = 20; ion source gas1 = 20; collision gas = 12; interface heater temperature = 150; entrance potential = 10; collision cell exit potential = 9; and Q1/Q3 = low resolution.

E. coli cell-free synthesis was performed using a Musaibou-Kun protein synthesis kit (Catalog #A183-0242, Taiyo Nippon Sanso Corporation, Tokyo, Japan). For ALACAT synthesis, an amino acid cocktail with lysine and arginine universally labeled with 13C and 15N (Catalog # A91-0128, Taiyo Nippon Sanso Corporation) was used. All synthetic reactions were performed using an Xpress micro-dialyzer MD100 with molecular weight cut-off of 12–14 kDa (Scienova, Spitzweidenweg, Germany) inserted into a 2 mL microtube. Before synthesis, 825 μL of the outer solution was mixed with 75 μL amino acid cocktail and 100 μL distilled water, incubated at 30 °C, and added to the outside of the dialysis unit at the start of synthesis. Then, 77.5 μL of the internal solution for synthesis was mixed with 10 μL template DNA (50 ng/μL), 7.5 μL amino acid cocktail, and 5 μL distilled water, and added to the dialysis unit. The synthesis reaction was carried out at 30 °C for 18 hours. After the synthesis was completed, all the solution in the dialysis unit was collected into a new tube.