Enrichment of Modified Peptides via Immunoaffinity Precipitation with Modification-Specific Antibodies

Immunoaffinity precipitation is an effective method of purifying select protein posttranslational modifications (PTMs) for proteomic analysis via mass spectrometry. Peptides containing a modification of interest are isolated directly from protease-digested cellular protein extracts using an antibody with specificity against the modification, and the modified peptides are analyzed by tandem mass spectrometry. Antibodies now exist with specificity for a variety of individual PTMs, such as phosphotyrosine, acetyl-lysine, methyl-arginine, ubiquitylation (i.e., diglycyl-lysine affinity), etc. Here we outline a generalized protocol for the purification of modified peptides by immunoaffinity precipitation. The main restriction for using this protocol is the availability of an antibody against the modification of interest. To purify modified peptides, antibodies are first conjugated to a solid support, such as agarose beads. The beads are then incubated with a complex peptide mixture, derived from a cellular lysate, under neutral pH to facilitate binding of modified peptides. The incubation time can vary from 30 min to overnight, depending upon the antibody used and the complexity of the peptide sample. Finally, acidic buffer conditions are used to elute the PTM-enriched bound peptides for mass spectrometry analysis.