Software advancements in the last several years have had a significant impact on proteomics from method development to data analysis. Herein, we detail a method, which uses our in-house developed software tool termed Skyline, for empirical refinement of candidate peptides from targeted proteins. The method consists of four main steps from generation of a testable hypothesis, method development, peptide refinement, to peptide validation. The ultimate goal is to identify the best performing peptide in terms of ionization efficiency, reproducibility, specificity, and chromatographic characteristics to monitor as a proxy for protein abundance. It is important to emphasize that this method allows the user to perform this refinement procedure in the sample matrix and organism of interest with the instrumentation available. Finally, the method is demonstrated in a case study to determine the best peptide to monitor the abundance of surfactant protein B in lung aspirates.
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