Part I: characterization of the extracellular proteome of the extreme thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS2

Abstract

The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS(2) analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C(18) bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C(18) bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties.

Fig. 1

Initial sample preparation employing three frequently used protein purification techniques and corresponding 1D gel images. a Schematic of acetone precipitation, trichloroacetic acid precipitation, and phenol extraction methods as performed on 50 μg secretome samples. b and c 1D SDS-PAGE experiments of the purified protein samples visualizing with Bio-Safe Coomassie and Sypro Ruby, respectively. The numbered lanes correspond to the numbered purification methods. * represents the control sample in which the secretome aliquot was not processed prior to gel electrophoresis.

Fig. 2

Investigation of additional sample desalting and protein purification techniques evaluated by gel electrophoresis. a Schematic of drop dialysis, stationary phase beads, and molecular weight cut off filter methods. b 1D SDS-PAGE experiment of secretome sample preparation methods. Sample loss occurred during drop dialysis over 6 hours due to the drop exceeding the membrane area. The numbered lanes correspond to the numbered purification methods.

Fig. 3

Order of operation of sample processing techniques for optimal 1D SDS-PAGE experiment. The numbered lanes correspond to combinations of the described purification methods. The numbers below the gel indicate the number of proteins identified by each method as well as the corresponding percentage based on 71 uniquely identified proteins from the C. saccharolyticus secretome sample.

Fig. 4

Experimental workflow from start to finish of secretome sample preparation and evaluations. Order of operation investigations analyzed by GeLC-MS² were assessed through a bioinformatic platform consisting of Mascot, ProteoIQ, and prediction servers. The sample preparation technique identifying the most proteins in conclusion of database searching (black outlined files) was suggested for use in future studies. Complementing the protein identification and spectral counts, prediction servers offered insight into the probable biological location of each protein.