Table 3 Development and validation of a PRM assay to detect ovarian cancer protein biomarkers in serum
Experiments for method characterization | Digested matrix | Standard | Internal standard | Experimental design |
|---|---|---|---|---|
Response curve | Sera (1 µg on LC column) | Mixture of 64-light peptides; 0.375 to 4800 fmol | Mixture of 64-heavy peptides; 200 fmol/peptide | Each light + heavy peptide mix was spiked into digested matrix, followed by LC-PRM analysis (n = 3) on the same day. Top 3 transition ions were selected for quantification unless otherwise indicated. LOD was determined using the average plus 3 × standard deviation of the blank signal. LOQ = 3 × LOD |
Validation of repeatability | Sera (1 µg on LC column) | Mixture of 64-light peptides: 2x (low), 50x (medium), and 200x (high) LOQ | Mixture of 64-heavy peptides; 200 fmol/peptide | Each sample of 3 concentrations (low, medium, and high) was analyzed in triplicate on 5 different days |
Assessment of selectivity | 6 biological replicates of sera (1 µg on LC column) | Mixture of 64-light peptides: no spike (blank), 25 × LOQ, and 50 × LOQ | Mixture of 64-heavy peptides; 200 fmol/peptide | Six biological replicates of undepleted sera with spiked-in light and heavy peptides were analyzed in duplicate in the same experimental block |
Quantification of endogenous peptides | 69 patient sera specimens (20 µg on column) |  | Mixture of 64-heavy peptides; 200 fmol/peptide | Endogenous analytes with spiked-in heavy peptides were analyzed in duplicate |