Table 1.
Overview of validation and quantitation experiments
| CPTAC experiment | Outcome | Plasma samplea | Standard (fmol on LC column for each injection)b | Normalizer (fmol on LC column for each injection)b | Experimental designc |
|---|---|---|---|---|---|
| 1 | Response curve | Pooled (N = 30)d | SIS 20,000 to 0.16; dilution pattern: 1:10:10:5:2:2:2:2:2:2:2:2 | NAT 200 | Digested sample was spiked with serial dilutions of SIS peptides and analyzed on the same day |
| 2 | Validation of assay repeatability | Pooled (N = 30)d | SIS 2.5× and 5× (low), 50× (medium), and 500× (high) assay LLOQ | NAT 200 | Five aliquots of the same digested sample were spiked and analyzed on five different days |
| 3 | Assessment of assay selectivity | Individual (N = 6)d | NAT no spike, 50×, and 500× assay LLOQ | SIS 100× assay LLOQ | Six biological replicates of the digested matrix were spiked and analyzed on the same day. |
| 4 | Validation of assay stability | Pooled (N = 30)d | SIS 100× assay LLOQ | NAT 200 | Six aliquots of the digested and spiked sample were analyzed after being stored at 4 and −80 °C for various periods of time |
| 5 | Reproducible detection of the endogenous analyte | Pooled (N = 30d | – | SIS 100× assay LLOQ | Five aliquots of the same sample were digested, spiked, and analyzed on five different days |
| Quantitation | Reference range of target protein concentrations | Individual (N = 6 for each strain)e | – | SIS 100× assay LLOQ | Individual samples were digested, spiked, and analyzed. Concentration ranges of endogenous analytes were determined using a calibration curve prepared in a surrogate matrix |
SIS stable isotope labelled peptide, NAT natural (unlabelled) peptide, LLOQ lower limit of quantification
aN refers to a number of biological replicates
bIn all experiments except for the CPTAC Experiment 1, concentration-balanced mixtures of standards were used in order to reflect the dynamic range of the individual assays and the concentrations of the respective endogenous analytes in mouse plasma. Validated assay LLOQ values for each assay are listed in Supplementary Data 2
cSamples were analyzed using a single injection (Quantification), two injections (Experiment 4) or three injections (Experiments 1, 2, 3, and 5). According to the CPTAC guidelines, different days were defined as different calendar days with digestions separated by at least 16 h
dPlasma samples from C57BL/6 mice (BioReclamationIVT)
ePlasma samples from C57BL/6 mice (BioReclamationIVT), CD1, 129S1/SvlmJ, NOD/SCID/J#1303, Balb/cJ, C57BL/6/CRL, and C57BL/6J (Toronto Centre for Phenogenomics)