ExSTA: External Standard Addition Method for Accurate High‐Throughput Quantitation in Targeted Proteomics Experiments

PURPOSE: Targeted proteomics using MRM with stable‐isotope‐labeled internal‐standard (SIS) peptides is the current method of choice for protein quantitation in complex biological matrices. Better quantitation can be achieved with the internal standard‐addition method, where successive increments of synthesized natural form (NAT) of the endogenous analyte are added to each sample, a response curve is generated, and the endogenous concentration is determined at the x‐intercept. Internal NAT‐addition, however, requires multiple analyses of each sample, resulting in increased sample consumption and analysis time. EXPERIMENTAL DESIGN: To compare the following three methods, an MRM assay for 34 high‐to‐moderate abundance human plasma proteins is used: classical internal SIS‐addition, internal NAT‐addition, and external NAT‐addition—generated in buffer using NAT and SIS peptides. Using endogenous‐free chicken plasma, the accuracy is also evaluated. RESULTS: The internal NAT‐addition outperforms the other two in precision and accuracy. However, the curves derived by internal vs. external NAT‐addition differ by only ≈3.8% in slope, providing comparable accuracies and precision with good CV values. CONCLUSIONS AND CLINICAL RELEVANCE: While the internal NAT‐addition method may be “ideal”, this new external NAT‐addition can be used to determine the concentration of high‐to‐moderate abundance endogenous plasma proteins, providing a robust and cost‐effective alternative for clinical analyses or other high‐throughput applications.