Limited Proteolysis Mass Spectrometry to Identify Protein Structural Differences in Brain Tissue

Structural proteomics methods allow for the proteome-wide interrogation of protein structural differences between two different conditions. Limited proteolysis mass spectrometry (LiP-MS), as originally implemented by the Picotti lab, utilizes a promiscuous protease to cleave at solvent-exposed regions of a protein to encode structural information, which is then read out with mass spectrometry proteomics. Here, we present a protocol that details experimental steps and data analysis for a LiP-MS workflow. First, tissue is homogenized under native conditions and then subjected to limited proteolysis using proteinase K (PK). The samples are prepared for mass spectrometry, and data are acquired using either data-dependent acquisition (DDA) or data-independent acquisition (DIA). Raw data is processed using FragPipe, and raw ion abundances are processed in FragPipe Limited-Proteolysis Processor (FLiPPR). Proteins with structural changes between the two conditions are identified in a proteome-wide manner. Key features • Protocol describes how to perform limited proteolysis mass spectrometry to identify proteins in brain tissue with structural changes proteome-wide between two experimental conditions. • Includes context for how to ensure results are reliable, using permutation analyses. • Utilizes tools (FragPipe and FLiPPR) that are free and open source. • Sample preparation can be performed in two days, not including mass spec acquisition and data analysis.