Figure 3.

Two-dimensional representations of the quantitative biases by (A) peptide-, extended-peptide-, and protein-level calibration approaches and (B) the two “hybrid” calibration approaches (c.f., Figure 1). QC samples were prepared by spiking blank plasma with pure protein at three levels: 1.6, 10, and 80 μg/mL. The purities of all standards were accurately measured by the quantitative amino acid analysis method to eliminate bias arising from possible inaccurate purity. Five aliquots of each QC sample were individually prepared and analyzed in replicates by the five calibration approaches. Each sample was analyzed three times in each of two different days (day 1 and day 14, N = 6, shown as individual data points). For every calibration method, the quantitative values were obtained independently using the two signature peptides (SP) (i.e., the GPS and TVA peptides). The two axes represent the quantitative biases by the two SP. The red box in the center of each panel denotes the zone of <20% bias, while the golden box signifies the zone of <10% bias.