Experimental approach for deep proteome measurements from small-scale microbial biomass samples.
Many methods of microbial proteome characterizations require large quantities of cellular biomass (>1-2 g) for sample preparation and protein identification. Our experimental approach differs from traditional techniques by providing the ability to identify the proteomic state of a microbe from a few milligrams of starting cellular material. The small-scale, guanidine lysis method minimizes sample loss by achieving cellular lysis and protein digestion in a single-tube experiment. For this experimental approach, the freshwater microbe Shewanella oneidensis MR-1 and the purple non-sulfur bacterium Rhodopseudomonas palustris CGA0010 were used as model organisms for technology development and evaluation. A 2-D LC-MS/MS comparison between a standard sonication lysis method and the small-scale guanidine lysis techniques demonstrates that the guanidine lysis method is more efficient with smaller sample amounts of cell pellet (i.e., down to 1 mg). The described methodology enables deeper proteome measurements from a few milliliters of confluent bacterial cultures. We also report a new protocol for efficient lysis from small amounts of natural biofilm samples for deep proteome measurements, which should greatly enhance the emerging field of environmental microbial community proteomics. This straightforward sample boiling protocol is complementary to the small-scale guanidine lysis technique, is amenable for small sample quantities, and requires no special reagents that might complicate the MS measurements.
Thompson
Melissa R
MR
Graduate School of Genome Science and Technology, Oak Ridge National Laboratory-University of Tennessee, Knoxville, Tennessee 37830, USA.
Chourey
Karuna
K
Froelich
Jennifer M
JM
Erickson
Brian K
BK
VerBerkmoes
Nathan C
NC
Hettich
Robert L
RL
D001426:Bacterial Proteins / Q000378:metabolism*; D018533:Biomass; D019295:Computational Biology; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*; D012241:Rhodopseudomonas / Q000378:metabolism*; D020592:Shewanella / Q000378:metabolism*
D001426:Bacterial Proteins; D020543:Proteome