Defining elastic fiber interactions by molecular fishing: an affinity purification and mass spectrometry approach.
Deciphering interacting networks of the extracellular matrix is a major challenge. We describe an affinity purification and mass spectrometry strategy that has provided new insights into the molecular interactions of elastic fibers, essential extracellular assemblies that provide elastic recoil in dynamic tissues. Using cell culture models, we defined primary and secondary elastic fiber interaction networks by identifying molecular interactions with the elastic fiber molecules fibrillin-1, MAGP-1, fibulin-5, and lysyl oxidase. The sensitivity and validity of our method was confirmed by identification of known interactions with the bait proteins. Our study revealed novel extracellular protein interactions with elastic fiber molecules and delineated secondary interacting networks with fibronectin and heparan sulfate-associated molecules. This strategy is a novel approach to define the macromolecular interactions that sustain complex extracellular matrix assemblies and to gain insights into how they are integrated into their surrounding matrix.
Cain
Stuart A
SA
Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M139PT, United Kingdom. stuart.a.cain@manchester.ac.uk
McGovern
Amanda
A
Small
Elaine
E
Ward
Lyle J
LJ
Baldock
Clair
C
Shuttleworth
Adrian
A
Kielty
Cay M
CM
D002135:Calcium-Binding Proteins / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004547:Elastic Tissue / Q000378:metabolism*; D000071838:Fibrillin-1; D000071837:Fibrillins; D006493:Heparin / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D000077153:Progranulins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012984:Software; D054392:Adipokines
D002135:Calcium-Binding Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D006493:Heparin; D036341:Intercellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D010455:Peptides; D000077153:Progranulins; D011994:Recombinant Proteins; C503225:CLSTN1 protein, human; C000605722:FBN1 protein, human; D054392:Adipokines; C492314:GRN protein, human