1a.Objectives (from AD-416):
Identify and characterize differentially expressed serum proteins and peptides associated with poultry metabolic skeletal problems such as femoral head separation (FHS) and tibial dyschondroplasia (TD).
1b.Approach (from AD-416):
Sera collected from FHS and TD-affected chickens, along with normal sera, will be analyzed to identify differentially occurring proteins and peptides. Low abundance proteins will be enriched while high abundance will be depleted using a library of hexapeptide-based affinity matrix called ProteoMiner. The proteins will be analyzed by 2D gel electrophoresis and characterized by peptide mass fingerprints and tandem mass spectrometry. The peptides will be enriched by solid phase extraction on C18 magnetic beads and analyzed by matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry. The characterization of the peptides will be done using tandem mass spectrometry. The identification of both proteins and peptides will be done using MASCOT and/or other similar data base search.
Poultry leg problems in young birds are primarily associated with leg joint disorders such as tibial dyschondroplasia (TD) and femoral head necrosis. Some of these problems can be experimentally induced. Since cartilage is the target organ affected, we reasoned that studying the changes in chondrocyte under experimental condition may provide clues to possible changes in those metabolites in the blood. Hence, we treated chondrocytes with thiram that produces TD and monitored the proteomic changes in the cells. Our results show that certain heat shock protein (HSP) factors such as HSP 70 and HSP 47 are affected by thiram, suggesting that these stress-induced factors may be biochemical portals for some of those problems.
We developed methods by chemical extraction followed by ion exchange and reverse phase chromatography and monitored peptides by HPLC-electrospray ionization mass spectroscopy. Our comparative study using serum from control and lame broilers showed differential changes in several peptides. We are in the process of characterizing those peptides by mass spectrometry and in the process of understanding their significance.