Fig. 1

Overall strategy for building block assembly of short and long ALACATs. The smallest unit of an ALACAT is a single double-stranded oligonucleotide encoding peptides (one, two, more) for quantification of a single target protein, flanked on either side by tripeptides that preserve the natural primary sequence context. These oligonucleotides include linker regions compatible with a Type IIS restriction enzyme (BsaI) that allows all five Qbricks to be assembled in the correct order in a single ligation reaction (a, odd cycle assembly) to form short ALACATs. In turn, these short ALACATs contain DNA sequences that are compatible with a second Type IIS restriction enzyme (SapI) and can be similarly assembled in a one tube reaction to create a long ALACAT (b, even cycle assembly). The vectors for the odd and even cycles both include in-frame fusions to glu-fibrinopeptide and c-myc encoding regions (quantification) and a hexahistidine tag (purification)