Targeted Mass Spectrometry Analysis of Protein Phosphorylation by Selected Ion Monitoring Coupled to Parallel Reaction Monitoring (tSIM/PRM).

Recent developments in targeted mass spectrometry-based proteomics have provided new methodological solutions for accurate and quantitative analysis of proteins and their posttranslational control, which has significantly advanced our understanding of stress responses in different plant species. Instrumentation allowing high-resolution, accurate-mass (HR/AM) analysis has provided new acquisition strategies for targeted quantitative proteomic analysis by targeted selected ion monitoring (tSIM) and parallel reaction monitoring (PRM). Here we report a sensitive and accurate method for targeted analysis of protein phosphorylation by tSIM coupled to PRM (tSIM/PRM). The tSIM/PRM method takes advantage of HR/AM mass spectrometers and benefits from the combination of highly sensitive precursor ion quantification by tSIM and highly confident peptide identification by spectral library matching in PRM. The detailed protocol describes tSIM/PRM analysis of Arabidopsis thaliana foliar proteins, from the building of a spectral library to sample preparation, mass spectrometry, and data analysis, and provides a methodological approach for specifying the molecular mechanisms of interest.