Analysis of the bovine plasma proteome by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry
In this study, the bovine plasma proteome was analysed using a three step protocol: (1) plasma was treated with a combinatorial peptide ligand library (CPLL) to assimilate the differences in concentrations of different proteins in raw plasma; (2) CPLL-treated material was fractionated by three standard electrophoretic separation techniques, and (3) samples were analysed by nano-liquid chromatography (nLC) matrix-assisted laser desorption/ionisation (MALDI) time-of-flight tandem (TOF/TOF) mass spectrometry. The efficiencies of three fractionation protocols for plasma proteome analysis were compared. After size fractionation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), resolution of proteins was better and yields of identified proteins were higher than after charge-based fractionation by preparative gel-free isoelectric focussing. For proteins with isoelectric points >6 and molecular weights ⩾63 kDa, the best results were obtained with a ‘shotgun’ approach, in which the CPLL-treated plasma was digested and the peptides, rather than the proteins, were fractionated by gel-free isoelectric focussing. However, the three fractionation techniques were largely complementary, since only about one-third of the proteome was identified by each approach.