uploading the compiled list of protein spectral counts generated by Scaffold software.
The validity of the current global rhesus monkey tissue proteomics data, identified by the human alternative database method, was evaluated by cluster analysis, western blot analysis and immunohistochemistry. As shown in Fig 5A, hierarchical analysis provided a clustering tree view informing physiological relevance between monkey tissues. Gene and hierarchical analysis were performed using TreeView (v1.60) software provided by Eisen Lab (http://rana. lbl.gov/EisenSoftware.htm). Briefly, a combined list of protein accession numbers and corresponding spectral counts (as identified from multiple organs) was loaded into the Gene Cluster Software (Eisen et al., Stanford University, USA) to generate a TreeView data file for further analysis[27]. From the cluster analysis of female monkey tissues, ovary, cerebellum and liver showed high relevance with breast, frontal cortex and pancreas respectively, which are considered to have similar physiological functions. Two-dimensional hierarchical analysis also demonstrated that tissues with high relevance showed similar distributions and intensities of protein components. MSLN (mesenteric lymph 
node) and PBD (proximal bile duct) were displayed as tissues with high similarity, which was due to high intensities of structural proteins (e.g. cytokeratins, actin filaments), which are known to be representative proteins in smooth muscle tissues. Additionally, differential protein expression exhibited as spectral counts were confirmed by western blot analysis. Target proteins were chosen on the basis of differential expression when comparing their raw spectral counts given by Scaffold analysis of MS data. Fig 5B shows the altered expression of vimentin, -actin, GAPDH, -catenin and HSP-70 in female tissue samples. This data corresponds well with the spectral count data in most cases. The proteomic data was also confirmed by visualizing the expression level of vimentin from the organ tissues using immunohistochemistry (IHC). Fig 5C is showing the images of vimentin expression from various organ tissues from male and female subjects. Male mesenteric lymph node tissue was used as a control since it provided high spectral counts (5C, lower right). IHC images are in good agreement with proteomic dataset, thus confirming 483367-10-8 higher expression for vimentin in proximal bile duct, mesenteric lymph node, and in the reproductive organ tissues.
Validation of proteomic datasets. (A) Heat map provided by cluster analysis of proteomic dataset from female monkey indicating the distribution and relative abundances of identified proteins. Also, the clustered organs are presented as a tree view (B) Representative western blot images of common proteins identified from female organs (1, Frontal cortex; 2, cerebellum; 3, mesenteric lymph node; 4, liver; 5, pancreas; 6, proximal bile duct; 7, breast; 8, ovary; 9, clitoris). Band intensities corresponding to their raw spectral counts provided by Scaffold software (C) Immunohistochemistry images (lower) with H&E staining (upper) of monkey organ tissues presenting different expression levels of vimentin. Mesenteric lymph node tissue from male subject was used as a negative control (lower, left).
We conclude that the alternative human database search of LC-MS/MS data is a simple and powerful strategy to study large-scale, global proteomics of non-human primate animal models having currently incomplete pro
