Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach

Journal Article · Tue Jan 01 04:00:00 EST 2013 · Analytical Chemistry

DOI:https://doi.org/10.1021/ac3032838· OSTI ID:1088121

Dykstra, Andrew B ^[1]; Raman, Babu ^[2]; Cook, Kelsey ^[1]

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1. ORNL
2. Dow Chemical Company, The

Post-translational modifications (PTMs) are known to play a significant role in many biological functions. The focus of this study is to characterize the post-translational modifications of the cellulosome protein complex used by the bacterium Clostridium thermocellum to better understand how this protein machine is tuned for enzymatic cellulose solubilization. To enhance comprehensive characterization, the extracellular cellulosome proteins were analyzed using multiple proteolytic digests (trypsin, Lys-C, Glu-C) and multiple fragmentation techniques (collisionally-activated dissociation, electron transfer dissociation, decision tree). As expected, peptide and protein identifications were increased by utilizing alternate proteases and fragmentation methods, in addition to the increase in protein sequence coverage. The complementarity of these experiments also allowed for a global exploration of PTMs associated with the cellulosome based upon a set of defined PTMs that included methylation, oxidation, acetylation, phosphorylation, and signal peptide cleavage. In these experiments, 85 modified peptides corresponding to 28 cellulosome proteins were identified. Many of these modifications were located in active cellulolytic or structural domains of the cellulosome proteins, suggesting a level of possible regulatory control of protein function in various cellulotyic conditions. The use of multiple enzymes and fragmentation technologies allowed for independent verification of PTMs in different experiments, thus leading to increased confidence in PTM identifications.

• Research Organization: Oak Ridge National Laboratory (ORNL)
• Sponsoring Organization: SC USDOE - Office of Science (SC)
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 1088121
• Journal Information: Analytical Chemistry, Journal Name: Analytical Chemistry Journal Issue: 6 Vol. 85; ISSN 0003-2700
• Country of Publication: United States
• Language: English