Data Independent Acquisition to Inform the Development of Targeted Proteomics Assays Using a Triple Quadrupole Mass Spectrometer

Mass spectrometry based targeted proteomics methods provide sensitive and high-throughput analysis of selected proteins. To develop a targeted bottom-up proteomics assay, peptides must be evaluated as proxies for the measurement of a protein or proteoform in a biological matrix. Candidate peptide selection typically relies on predetermined biochemical properties, data from semi-stochastic sampling, or by empirical measurements. These strategies require extensive testing and method refinement due to the difficulties associated with prediction of peptide response in the biological matrix of interest. Gas-phase fractionated (GPF) narrow window data-independent acquisition (DIA) aids in the development of reproducible selected reaction monitoring (SRM) assays by providing matrix-specific information on peptide detectability and quantification by mass spectrometry. To demonstrate the suitability of DIA data for selecting peptide targets, we reimplement a portion of an existing assay to measure 98 Alzheimers disease proteins in cerebrospinal fluid (CSF). Peptides were selected from GPF-DIA based on signal intensity and reproducibility. The resulting SRM assay exhibits similar quantitative precision to published data, despite the inclusion of different peptides between the assays. This workflow enables development of new assays without additional up-front data acquisition, demonstrated here through generation of a separate assay for an unrelated set of proteins in CSF from the same dataset.