Fig. 2
From: Emerging roles of non-histone protein crotonylation in biomedicine
source before entering the mass spectrometer. MS and MS/MS spectra are then computationally processed to deduce peptide sequences, including the presence and location of crotonylation, and to quantify the abundance of crotonylated peptides and proteins
Schematic diagram of the experimental procedures for mass spectrometry-based global analysis. Proteins are extracted from cells or tissues and digested into peptides with a protease such as trypsin. The tryptic peptides are then separated and fractionated by high pH reverse-phase high performance liquid chromatography (HPLC). Proteolysis of whole-cell protein extracts generates numerous peptides, but only a small fraction is crotonylated. To enrich lysine crotonylated peptides, pan-Kcr antibodies are applied to identify the crotonylated peptides in complex peptide mixtures using immunoaffinity purification. The resulting peptides are ionized in the electrospray