Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin beta 1

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin beta 1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin beta 1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin beta 1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin beta 1, alpha 4 and alpha 6 or beta 1, alpha 6, alpha 2, and alpha 5, respectively.