PMC

Distribution of peptide response and relation to assay performance. A, Histogram of peptide response measured by LC-MRM-MS for 216 peptides. Responses vary over three orders of magnitude. B, Distribution of peptide response (i.e. intensity) as a function of assay grade.

Generation of antipeptide antibodies using a multiplexed peptide immunization procedure. A, Distribution across protein targets of the number of peptides that were used for multiplexed immunization. B, Distribution across all proteins of the number of antibodies that were affinity purified. C, Summary of the distribution of number of antibodies that were affinity purified for each immunization multiplex level. D, Distribution of the yields of affinity-purified polyclonal antibodies across all peptides.

An example assay characterization report for the peptide YLAPSGPSGTLK from the protein FSCN1. A, Response curves for each of three monitored transitions. B, Example chromatograms for each transition at two concentrations (0.05 fmol/μl and 0.5 fmol/μl heavy peptide; 10 fmol/μl light peptide). Traces show the chromatograms for the (heavy) stable isotope-labeled peptide (in blue) overlaid on traces of the (light) peptide (in pink).

Distribution of detection levels for graded assays. Distributions of the detection levels are plotted for each group of graded assays (A–D). Detection levels (plotted on log₂ scale) are estimations of assay sensitivity determined from four-point response curves as part of assay characterization. Calculation of protein concentration assumes enrichment from 10 μl plasma and equal stoichiometry between peptide and protein (i.e. complete trypsin digestion).

Study overview. The overall goals of the study were to determine the success rate of generating SISCAPA assays and evaluate the assay development pipeline. Multiple proteotypic peptides were identified for each targeted protein and used as immunogens to generate affinity purified antipeptide polyclonal antibodies. Development of the SISCAPA assays included performing quality control and MRM method development for each targeted peptide. The resulting SISCAPA assays were screened in plasma using a response curve and the results analyzed to determine the overall success rate and performance of the assays.