iTRAQ Manatee

In 2013, two large-scale Florida manatee (Trichechus manatus latirostris) mortality episodes were reported on separate coasts of Florida. The east coast mortality episode was associated with an unknown etiology in the Indian River Lagoon (IRL). The west coast mortality episode was attributed to a persistent Karenia brevis algal bloom or ‘red tide’ centered in Southwest Florida. To investigate these two mortality episodes, proteomic experiments using two-dimensional difference gel electrophoresis (2D-DIGE) followed by protein identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS) were conducted, along with a separate gel-free analysis using isobaric tags for relative and absolute quantification (iTRAQ) LC-MS/MS. In comparison to the control group, manatees from the IRL, an area associated with an unknown mortality episode, displayed increased levels of several proteins in their serum samples. These increased proteins, which were identified in the iTRAQ experiment, included kininogen-1 isoform 1 (average ratio 1.38), protein AMBP (1.38), histidine-rich glycoprotein (1.34), properdin (1.30), and complement C4-A isoform 1 (1.25). In the red tide group, ceruloplasmin (2.32), pyruvate kinase isozymes M1/M2 isoform 3 (2.29), angiotensinogen (2.08), complement C4-A isoform 1 (1.83), and complement C3 (1.42) were increased. The proteins kininogen-1 isoform 1, histidine-rich glycoprotein, complement C4-A isoform 1, angiotensinogen, and complement C3 were also identified in increased levels in the 2D-DIGE experiment (Table 2b). These proteins are associated with acute-phase response, amyloid formation and accumulation, copper and iron homeostasis, the complement cascade pathway, and other important cellular functions. The increased level of complement C4 protein observed in both the red tide and unknown mortality episode groups was confirmed through the use of Western Blot.