Fig. 1

Steps in SRM method development. I ANG II-regulated proteins most likely to be found in urine were selected for SRM method development. Most highly observable proteotypic (unique) peptides were selected from Peptide Atlas, and searched with the protein BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to ensure their uniqueness. Transitions were then selected by in silico digestion in Skyline. II To develop SRM methods we purchased 26 crude, synthetic, unlabeled peptides, which were used to determine retention time and order of transitions. 11 additional peptides had methods previously developed in human primary cells. III To determine reproducibility and recovery of sample preparation, we spiked 1 µg of BSA protein into urine containing 100 µg of total protein. We examined four different sample preparation protocols: ACN precipitation, Amicon filter concentration, digestion with trypsin alone or digestion with Lys-C and trypsin. Finally we spiked in three heavy isotope-labeled peptides corresponding to BSA, and fractions containing 20 µg total protein were subjected to C₁₈ microextraction, prior to injection on triple quadrupole