Matrix-matched calibration curves

Mass spectrometry is a powerful tool for quantifying protein abundance in complex samples. Advances in sample preparation and the development of data independent acquisition (DIA) mass spectrometry approaches have increased the number of peptides and proteins measured per sample. However, methods to assess quantitative figures of merit (e.g. lower limit of quantification, LLOQ) are not easily extended to multiplex assays with hundreds or thousands of analytes. Here we present a series of experiments demonstrating how to assess whether a peptide measurement is quantitative by mass spectrometry. In a study of the yeast proteome, only 52% of detected proteins (41% of detected peptides) have a peptide that is above the LLOQ in the reference material. A similar trend was observed in human cerebrospinal fluid, suggesting that this observation is not sample-specific. Our results demonstrate that increasing the number of detected peptides and proteins in an unbiased proteomics experiment does not necessarily result in increased numbers of peptides or proteins that can be measured quantitatively. Furthermore, our method provides an approach to determining useful figures of merit for hundreds to thousands of proteins in the same experiment.