Surface enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry has facilitated the discovery of disease specific protein profiles from different biological samples, such as serum and tissues, in a variety of diseases of man. These results have raised the possibility that protein profiles may become a powerful diagnostic tool and may be applicable to Ovine Johne's Disease (OJD).

In the first phase of this study, SELDI-TOF MS experiments have been rigorously optimized by using sheep serum applied on four different ProteinChip® Array surfaces, in combination with nine different binding/washing buffers and five different sample dilutions. The reproducibility study showed the range for mean within-chip coefficient of variation for peak intensity determined from up to 18 peaks using sera from 8 sheep was 13% - 18% and for mass accuracy was 0.01% - 0.02%. Corresponding values between-chip were 13% - 23% and 0.02% - 0.03% respectively.

Based on the results of the optimization experiment, a large scale biomarker discovery experiment has been conducted. We used SELDI-TOF MS to identify potential proteomic biomarkers from sheep serum that can differentiate between sheep infected with Mycobacterium avium subsp. paratuberculosis, uninfected sheep and those previously vaccinated with Gudair™. Univariate and two independent multivariate data analysis procedures: Linear Discriminant Analysis (LDA) and Classification and Regression Decision Tree (CART), have been used to develop classification models between contrasting populations. A panel of key polypeptides has been selected using both models for identification and further analysis.

To identify the serum proteins found using SELDI-TOF MS, a number of chromatographic procedures, include gel filtration, affinity and ion exchange chromatography are being used in a protein purification scheme. A 13.6 kDa protein, transthyretin, which was down-regulated in both infected and vaccinated serum samples, has been identified by MS/MS.