Proteomic Study of Ribosomal Proteins from Escherichia coli, Saccharomyces cerevisiae, Bos taurus, Gallus gallus, and Oncorhynchus tshawytscha: Application in a Teaching Laboratory Setting

Ribosomes are central to protein synthesis and our understanding of ribosomes has advanced antibiotics research. The proteomic study of ribosomes presented here utilizes a combination of differential centrifugation and matrix assisted laser desorption�/�ionization�-�time of flight mass spectrometry (MALDI�-TOFMS) to analyze ribosomes from various species in a teaching laboratory setting. Five biologically varied species were used: Escherichia coli (bacteria), Saccharomyces cerevisiae (yeast), Bos taurus (cow), Gallus gallus (chicken), and Oncorhynchus tshawytscha (Chinook salmon). Samples were lysed, ribosomes were isolated via ultracentrifugation using a discontinuous sucrose gradient and the individual protein subunits were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis. Tryptic digest and MALDI-TOFMS were then conducted on fifteen bands excised from the gel, and the mass spectra of both the whole protein sample and peptides were analyzed. Five out of these fifteen bands were positively identified as various ribosomal proteins, with two uncertain identifications. Additionally, three of the five positively identified proteins that travelled the same distance on the gel were determined to be orthologous. Finally, a class of 14 Biochemistry II students utilized these protocols, identified three ribosomal proteins and provided their evaluations of the ultracentrifugation-proteomics teaching laboratory.