Table 1 Overview of validation and quantitation experiments

CPTAC experiment	Outcome	Plasma sample^a	Standard (fmol on LC column for each injection)^b	Normalizer (fmol on LC column for each injection)^b	Experimental design^c
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 = 30^d	–	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)

Search