title: Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate.
abstract: Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.
authors.affiliation: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Roisin
authors.initials: R
authors.lastname: O'Connor
authors.forename: Lorna M
authors.initials: LM
authors.lastname: Cryan
authors.forename: Kieran
authors.initials: K
authors.lastname: Wynne
authors.forename: Andreas
authors.initials: A
authors.lastname: de Stefani
authors.forename: Desmond
authors.initials: D
authors.lastname: Fitzgerald
authors.forename: Colm
authors.initials: C
authors.lastname: O'Brien
authors.forename: Gerard
authors.initials: G
authors.lastname: Cagney
mesh_terms: D001792:Blood Platelets / Q000737:chemistry* / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D002465:Cell Movement; D002852:Chromatography, Ion Exchange; D045424:Complex Mixtures / Q000032:analysis*; D006801:Humans; D009000:Monocytes / Q000378:metabolism; D009928:Organ Specificity; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D001798:Blood Proteins; D045424:Complex Mixtures