Proteome-Wide Multipoint Internal Calibration Curves for Evaluating Peptide-Level Linearity in Relative Quantitative Proteomics

Mass spectrometry (MS)-based proteomics is known for its high accuracy in quantifying peptides and proteins using various calibration strategies including internal and external calibration curves. While external multipoint calibration curves are created from serial dilutions, they often fail to account for sample-specific matrix effects. In contrast, internal calibration curves account for the sample matrix but face scalability and cost challenges for whole proteome analyses. In this manuscript, we present a novel TMT-based multipoint internal calibration curve strategy, which enables the generation of internal calibration curves for all peptides identified within a proteome in a single experiment to assess their linearity prior relative quantification. We applied this strategy to human ovarian cancer cells to evaluate the linear quantitative responses of all of the identified peptides and reveal the significant proteome changes associated with cisplatin treatment.