Technical Abstract: Carotenoid-protein complex separation by size exclusion chromatography, protein fractionation by SDS-PAGE, and shotgun PROTEOMICS technology were used to identify and characterize carotenoid associated proteins (CAPs) of chromoplast-enriched suspensions from cassava intense yellow storage root. A non-denatured carotenoid-protein complex was separated in fractions 33-49 (peak 1) and a non-carotenoid-associated proteins in fractions 108-122 (peak 2). Proteins from each peak were separated by SDS-PAGE and in gel tryptic digested. Peptide sequences were directly obtained from the LC_MS/MS spectrum and peptide mass analyzed using MASCOT search engine. Protein identification was established using public sequence databases, and enhancement of confidence using four cassava EST databases from CIAT, EMBRAPA, ESTIMA, and RIKEN that were translated in all six reading frames and annotated to the Arabidopsis and Manihot database. Sequences from peak 1 and 2 identified 83 and 106 peptides, respectively, which matched to 26 and 39 known proteins names. Small Heat Shock Proteins (sHSPs) were the most abundant proteins present in the carotenoid-protein complex. Pfam domain analysis indicated three families of HSPs present in the carotenoid-protein complex with a variety of putative functionality. Western blot analysis recognized the presence of Fibrillin and Or_proteins in chromoplast enriched suspension and peak 2 of intense yellow root but not in peak 1 and white root. Performed qRT_PCR identified transcript abundance of four proteins identified in peaks 1 and 2 together with Fibrillin and Or_proteins indicating that all of them are highly expressed in intense yellow root. Transcript for CAP4 protein indicated the presence of a heteroduplex DNA strand distinct in the white and intense yellow phenotype, as judged by their dissociation curve from the qRT_PCR result and PCR product sequence. Cumulatively, these results indicate that sHSP play a important role in sequestration mechanism of carotenoids in cassava storage root, support the correlation between total carotenoid and protein content in the root tissue of pigmented cassava, and may provide new approaches for increasing nutritional value of domesticated cassava.