Quantitation of the phosphoproteome using the library-assisted extracted ion chromatogram (LAXIC) strategy.

Phosphorylation is a key posttranslational modification that regulates many signaling pathways, but quantifying changes in phosphorylation between samples can be challenging due to its low stoichiometry within cells. We have introduced a mass spectrometry-based label-free quantitation strategy termed LAXIC for the analysis of the phosphoproteome. This method uses a spiked-in synthetic peptide library designed to elute across the entire chromatogram for local normalization of phosphopeptides within complex samples. Normalization of phosphopeptides by library peptides that co-elute within a small time frame accounts for fluctuating ion suppression effects, allowing more accurate quantitation even when LC-MS performance varies. Here we explain the premise of LAXIC, the design of a suitable peptide library, and how the LAXIC algorithm can be implemented with software developed in-house.