Proteomics Strategy for Identifying Candidate Bioactive Proteins in Complex Mixtures: Application to the Platelet Releasate

Figure 1

Strategy for linking function and protein identity in biofluids. An off-line MuDPIT strategy was used to identify biologically active proteins from a complex mixture. Proteins released from platelets were separated by SCX/SAX chromatography. The collected fractions were analyzed for pro-migratory activity using a monocyte Boyden chamber assay. The fractions were separated in the second dimension by SDS-PAGE, and gel lanes were excised and the proteins were digested with trypsin. The resulting peptides were separated by RP-HPLC, ionized by electrospray, and subjected to MS/MS to identify potential pro-migratory proteins.