Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags

Abstract

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Fig 1.

Two-dimensional display of a capillary LC-FTICR analysis in which >50,000 putative polypeptides were detected from a tryptic digest of D. radiodurans proteins harvested in mid-log phase (OD₆₀₀ 0.3–0.4; 30°C). Inset shows portions of displays for peptides from D. radiodurans harvested in mid-log (Left), late-log (Center), and poststationary (Right) phases (spot size reflects relative abundance). Each individual spot corresponds to a peptide that can be identified along with the parent protein using AMTs. Spot identifications: 1: DR1314 (hypothetical protein); 2: DR1790 (homolog of yellow/royal jelly protein of insects); 3: DR1314 (hypothetical protein); 4: DR0309 (elongation factor Tu); 5: DR2577 (S-layer protein); 6: DR0989 (membrane protein); and 7: DR1124 (S-layer protein).

Fig 2.

(A) The qualitative pattern of ORF expression detected using AMTs for various conditions by TIGR assigned functional category (2) (red, AMTs detected; green, AMTs not observed). Each culture condition (as described in Experimental Protocols) was analyzed at least two times. (B) Expansion of the pattern for predicted hypothetical proteins, illustrating similarities and differences under different culture conditions. (C) Expansion showing several representative hypothetical proteins.

Fig 3.

Change in relative abundance of (•) RecA and (♦) DNA-directed RNA polymerase in cells after exposure to 17.5 kGy of ionizing radiation obtained be analysis of a mixture of cells grown on unlabeled media and the ¹⁵N-labeled reference proteome (control, nonirradiated cells). Cells were prepared at 0, 3, 7, 9, and 12 hr after exposure.