Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate.
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 acti...
| Publicado en: | Journal of Biomedicine & Biotechnology pp. 107859 - 107860 |
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| Autores principales: | , , , , , , |
| Formato: | research Journal Article |
| Publicado: |
Wiley-Blackwell
2010
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=105081925&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 105081925 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 11107243 137K jtl: Journal of Biomedicine & Biotechnology issn: 11107243 maglogo: N pubinfo: dt: 2010 pid: 480 pub: Wiley-Blackwell place: Malden, Massachusetts artinfo: ui: 105081925 2010748443 10.1155/2010/107859 NLM20368775 PMC2846341 105081925 ppf: 107859 ppct: 1 formats: fmt: @attributes: type: P tig: atl: Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate. aug: au: O'Connor R Cryan LM Wynne K de Stefani A Fitzgerald D O'Brien C Cagney G affil: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin 4, Ireland. sug: subj: Blood Platelets Blood Proteins Drugs Analysis Proteomics Methods Blood Platelets Metabolism Blood Proteins Metabolism Blotting, Western Cell Physiology Chromatography, Ion Exchange Human Monocytes Metabolism Immunity Reproducibility of Results ab: 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. pubtype: Academic Journal doctype: research Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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