Sortase A-mediated site-specific immobilization of peptides and proteins for interactome analysis by LC-MS/MS

摘要

Affinity purification-mass spectrometry(AP-MS) experiments are being increasingly used to study protein complexes and to identify protein-protein interactions. However, identification and quantification of low-abundant interaction partners is frequently challenging because of the limited dynamic range of LC-MS/ MS. The bait protein or antibody is often the dominant protein complicating the detection of less abundant interaction partners. Covalently crosslinking the bait to the matrix may facilitate MS analysis. However, most common approaches lead to randomly oriented crosslinks which may cause loss of functionality of the bait protein. Here we made use of sortase A-catalyzed transpeptidation to site-specifically attach peptides and proteins to agarose matrices, subsequently used for interaction analysis.