Fig. 2.

Construction of a human plasma protein QconCAT using the ALACAT strategy. A series of Qbricks were designed, each encoding two peptides for each of 25 plasma proteins. Groups of five were assembled in an odd cycle reaction to short ALACATs (301-305) that were expressed in vitro using wheat germ lysate and purified by virtue of their hexahistidine tag. In addition, the short ALACATs were assembled in an even cycle reaction into a single long ALACAT that was also expressed and purified (a). Each of the ALACATs was then digested and analysed by LC-MSMS; all peptides were detected (c, infilled peptides are those visible by LC-MS/MS (b); different colours define the short ALACAT origins of the peptides in the long ALACAT). Using common peptides (glu fibrinopeptide and c-myc epitope) as normalisation controls, the peak areas for peptides in short ALACATs were compared to the areas of the same peptides in the long ALACATs (d)