Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin beta1.
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 beta1 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 beta1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin beta1 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 beta1, alpha4 and alpha6 or beta1, alpha6, alpha2, and alpha5, respectively.
Klockenbusch
Cordula
C
The Biomedical Research Centre, University of British Columbia, Vancouver, BC V6T1Z3, Canada.
Kast
Juergen
J
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D001792:Blood Platelets / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D047468:Immunoprecipitation; D019012:Integrin beta1 / Q000737:chemistry* / Q000378:metabolism; D019169:Jurkat Cells; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D011506:Proteins / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D003432:Cross-Linking Reagents; D019012:Integrin beta1; D046912:Multiprotein Complexes; D011506:Proteins; D005557:Formaldehyde