4 research outputs found
Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach
Post-translational modifications
(PTMs) are known to play a significant
role in many biological functions. The focus of this study is to optimize
an integrated experimental/informatics approach to more confidently
characterize the range of 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 complementary proteolytic
digestion/peptide fragmentation processes allowed for independent
verification of PTMs in different experiments, thus leading to increased
confidence in PTM identifications
Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach
Post-translational modifications
(PTMs) are known to play a significant
role in many biological functions. The focus of this study is to optimize
an integrated experimental/informatics approach to more confidently
characterize the range of 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 complementary proteolytic
digestion/peptide fragmentation processes allowed for independent
verification of PTMs in different experiments, thus leading to increased
confidence in PTM identifications
Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach
Post-translational modifications
(PTMs) are known to play a significant
role in many biological functions. The focus of this study is to optimize
an integrated experimental/informatics approach to more confidently
characterize the range of 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 complementary proteolytic
digestion/peptide fragmentation processes allowed for independent
verification of PTMs in different experiments, thus leading to increased
confidence in PTM identifications
Development of a Multipoint Quantitation Method to Simultaneously Measure Enzymatic and Structural Components of the <i>Clostridium thermocellum</i> Cellulosome Protein Complex
<i>Clostridium thermocellum</i> has emerged as a leading
bioenergy-relevant microbe due to its ability to solubilize cellulose
into carbohydrates, mediated by multicomponent membrane-attached complexes
termed cellulosomes. To probe microbial cellulose utilization rates,
it is desirable to be able to measure the concentrations of saccharolytic
enzymes and estimate the total amount of cellulosome present on a
mass basis. Current cellulase determination methodologies involve
labor-intensive purification procedures and only allow for indirect
determination of abundance. We have developed a method using multiple
reaction monitoring (MRM-MS) to simultaneously quantitate both enzymatic
and structural components of the cellulosome protein complex in samples
ranging in complexity from purified cellulosomes to whole cell lysates,
as an alternative to a previously developed enzyme-linked immunosorbent
assay (ELISA) method of cellulosome quantitation. The precision of
the cellulosome mass concentration in technical replicates is better
than 5% relative standard deviation for all samples, indicating high
precision for determination of the mass concentration of cellulosome
components