Type

Journal Article

Authors

Gerard Cagney
Colm O'Brien
Desmond Fitzgerald
Andreas de Stefani
Kieran Wynne
Lorna M Cryan
Roisin O'Connor

Subjects

Biochemistry

Topics
chemistry cell movement organ specificity proteomics metabolism blood proteins monocytes blood platelets humans blotting western reproducibility of results chromatography ion exchange complex mixtures

Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate. (2009)

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.
Collections Ireland -> University College Dublin -> PubMed

Full list of authors on original publication

Gerard Cagney, Colm O'Brien, Desmond Fitzgerald, Andreas de Stefani, Kieran Wynne, Lorna M Cryan, Roisin O'Connor

Experts in our system

1
Gerard Cagney
Royal College of Surgeons in Ireland
Total Publications: 54
 
2
Colm J O'Brien
University College Dublin
Total Publications: 54
 
3
D J Fitzgerald
Royal College of Surgeons in Ireland
Total Publications: 101
 
4
Kieran Wynne
Royal College of Surgeons in Ireland
Total Publications: 45