Targeted proteomics (parallel reaction monitoring) to quantify starch-biosynthetic proteins in yeast

Heterologous protein expression in Saccharomyces cerevisiae is a powerful testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. Here we modulated the expression each starch synthase together with a branching enzyme (derived from Arabidopsis thaliana) in a suite of yeast strains and quantified protein abundances using targeted proteomics. Specifically, we developed parallel reaction monitoring assays to quantify the Arabidopsis proteins SS1, SS2, SS3, SS4, BE2, BE3, ISA1, ISA2, among others (see Supplementary File 1, sheet 3) when expressed in yeast. We selected multiple surrogate peptides for each protein and characterized the assays with respect to their dynamic range (Supplementary Files 1 and 2). Data calibration was supported using crude stable isotope labeled (SIL) peptides.