N-Terminal Enrichment: Developing a Protocol to Detect Specific Proteolytic Fragments

摘要: Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, well regulating proteins in cancer; therefore, there is a significant need develop robust approaches for characterizing events. The current mass spectrometry (MS)-based proteomics techniques employs “bottom-up” strategy, which does not allow identification of different proteolytic since the strategy measures all small peptides from any given protein. aim this development enable effective specific fragments. protocol utilizes an acetylation reaction block N-termini protein, lysine residues. Following digestion, N-terminal enriched by removing that contain free amines, using amine-reactive silica-bond succinic anhydride beads. resulting sample has one peptide per reduces complexity allows increased analytical sensitivity compared global proteomics.1 We initially efficiency blocking acetic (a 42 Da modification) or propionic 56 our protocol. Both chemical reactions resulted comparable identifications ∼95 percent However, use allowed us distinguish vivo acetylated chemically-tagged peptides.2 In initial experiment mouse plasma, we were able identify >300 unique peptides, many known cleavage sites. This holds potential uncovering new information related pathways, will assist understanding about cancer biology efforts biomarkers various diseases.