Dmitry Shamshurin

Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression.

BackgroundClostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.ResultsRelative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.ConclusionsRelative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.

Effect of cyclization of N-terminal glutamine and carbamidomethyl-cysteine (residues) on the chromatographic behavior of peptides in reversed-phase chromatography

N-terminal loss of ammonia is a typical peptide modification chemical artifact observed in bottom-up proteomics experiments. It occurs both in vivo for N-terminal glutamine and in vitro following enzymatic cleavage for both N-terminal glutamine and cysteine alkylated with iodoacetamide. In addition to a mass change of -17.03 Da, modified peptides exhibit increased chromatographic retention in reversed-phase (RP) HPLC systems. The magnitude of this increase varies significantly depending on the peptide sequence and the chromatographic condition used. We have monitored these changes for extensive sets (more than 200 each) of tryptic Gln and Cys N-terminated species. Peptides were separated on 100 Å pore size C18 phases using identical acetonitrile gradient slopes with 3 different eluent compositions: 0.1% trifluoroacetic acid; 0.1% formic acid and 20 mM ammonium formate at pH 10 as ion-pairing modifiers. The observed effect of this modification on RP retention is the product of increased intrinsic hydrophobicity of the modified N-terminal residue, lowering or removing the effect of ion-pairing formation on the hydrophobicity of adjacent residues at acidic pHs; and possibly the increased formation of amphipathic helical structures when the positive charge is removed. Larger retention shifts were observed for Cys terminated peptides compared to Gln, and for smaller peptides. Also the size of the retention increase depends on the eluent conditions: pH 10≪trifluoroacetic acid

Quantitative proteomic analysis of the cellulolytic system of Clostridium termitidis CT1112 reveals distinct protein expression profiles upon growth on α-cellulose and cellobiose

UNLABELLED Clostridium termitidis CT1112 is an anaerobic, mesophilic, cellulolytic bacterium with potential applications in consolidated bioprocessing of lignocellulosic biomass. To understand how C. termitidis degrades lignocellulose, iTRAQ-based 2D HPLC-MS/MS proteomics was used to measure protein expression in cell lysates and extracellular (secretome) fractions of C. termitidis grown on α-cellulose and cellobiose at both exponential and stationary growth phases. Exoglucanases (GH48, GH9), endoglucanases (GH5, GH8, GH9), hemicellulases including xylanases (GH8, GH10, GH11, GH30) and mannanase (GH26) as well as extracellular adhesion proteins and cellulosome associated proteins, exhibited higher expression on cellulose-grown cells. The expression of these proteins increased with a decrease in growth rate. Non-cellulosomal proteins however did not change significantly between substrate conditions, although there were a few exceptions. Collectively, these would contribute to hydrolysis of lignocellulosic material for uptake through ABC sugar transport proteins. On cellobiose, chitinases (GH18) were expressed abundantly. Although a large number of proteins were shared between the fractions analyzed, some proteins were detected exclusively in the cellular fraction, while others were detected in the secretome. This study reports for the first time on the cellulolytic machinery employed by C. termitidis to hydrolyze cellulosic substrate and provides an understanding of how this microbe deconstructs biomass. BIOLOGICAL SIGNIFICANCE The genome of C. termitidis CT1112 contains genes for a wide variety of carbohydrate active enzymes. Based on bioinformatics analyses, many of these genes appear to encode cellulosome-associated proteins, while others may be secreted extracellularly. To understand how C. termitidis degrades and depolymerizes cellulosic substrates, cells were grown on simple and complex carbohydrates, and quantitative 4-plex iTRAQ-based 2D HPLC-MS/MS proteomics was applied to measure protein expression levels in biological replicates of both cell lysates and extracellular protein (secretome) fractions, at exponential and stationary phases of growth. The resulting data have provided insight into the range of substrates that may be hydrolyzed by C. termitidis, and may be useful in determining potential industrial applications of C. termitidis in biomass to bioenergy production via consolidated bioprocessing.

N-capping motifs promote interaction of amphipathic helical peptides with hydrophobic surfaces and drastically alter hydrophobicity values of individual amino acids.

Capping rules, which govern interactions of helical peptides with hydrophobic surfaces, were never established before due to lack of methods for the direct measurement of polypeptide structure on the interphase boundary. We employed proteomic techniques and peptide retention modeling in reversed-phase chromatography to generate a data set sufficient for amino acid population analysis at helix ends. We found that interactions of amphipathic helical peptides with a hydrophobic C18 phase are induced by a unique motif featuring hydrophobic residues in the N1 and N2 positions adjacent to the N-cap (Asn, Asp, Ser, Thr, Gly), followed by Glu, Gln, or Asp in position N3 to complete a capping box. A favorable N-capping arrangement prior to amphipathic helix may result in the highest hydrophobicity (retention on C18 columns) of Asp/Asn (or Glu/Gln) peptide analogues among all naturally occurring amino acids when placed in N-cap or N3 position, respectively. These results contradict all previously reported hydrophobicity scales and provide new insights into our understanding of the phenomenon of hydrophobic interactions.

The effect of various S-alkylating agents on the chromatographic behavior of cysteine-containing peptides in reversed-phase chromatography.

We investigate the influence of various alkylation chemistries on the reversed phase (RP) HPLC behavior of Cys-containing peptides under the most popular RP-HPLC conditions used in proteomics: C18 phases with trifluoroacetic acid (TFA) or formic acid (FA) as the ion pairing modifiers, and separation at pH 10. Akylating agents studied are iodoacetamide (IAM), iodoacetic acid (IAA), 4-vinylpyridine (4-VP), acrylamide (AA) and methyl methanethiosulfonate (MMTS). These were compared against the retention of identical peptides without alkylation, i.e. free cysteines. The intrinsic hydrophobicity values of the Cys residue under formic acid conditions for these modifications were found to increase in the following order: 4-VP<IAM<AA<IAA<free Cys<MMTS. The retention contribution of the positively charged 4-VP derivative is affected by the nature of the ion-pairing modifier; this is the most hydrophilic residue for formic acid based eluents, and second most hydrophilic behind IAM-alkylated Cys using TFA eluent. Switching to a basic condition dramatically decreases the retention of free cysteine and IAA-alkylated analytes due to the ionization of side-chains. The opposite effect is observed for 4-VP, which become neutral at basic pHs. The careful measurement of the hydrophobic contributions for these residues is vital to the development of accurate peptide retention prediction models; the incorporation of these modifications into our Sequence Specific Retention Calculator model is presented.

Transcriptomic and proteomic analyses of core metabolism in Clostridium termitidis CT1112 during growth on α-cellulose, xylan, cellobiose and xylose.

BackgroundClostridium termitidis CT1112 is an anaerobic, Gram-positive, mesophilic, spore-forming, cellulolytic bacterium, originally isolated from the gut of a wood feeding termite Nasusitermes lujae. It has the ability to hydrolyze both cellulose and hemicellulose, and ferment the degradation products to acetate, formate, ethanol, lactate, H2, and CO2. It is therefore ges in gene and gene product expression during growth of C. termitidis on cellobiose, xylose, xylan, and α–cellulose.ResultsCorrelation of transcriptome and proteome data with growth and fermentation profiles identified putative carbon-catabolism pathways in C. termitidis. The majority of the proteins associated with central metabolism were detected in high abundance. While major differences were not observed in gene and gene-product expression for enzymes associated with metabolic pathways under the different substrate conditions, xylulokinase and xylose isomerase of the pentose phosphate pathway were found to be highly up-regulated on five carbon sugars compared to hexoses. In addition, genes and gene-products associated with a variety of cellulosome and non-cellulosome associated CAZymes were found to be differentially expressed. Specifically, genes for cellulosomal enzymes and components were highly expressed on α–cellulose, while xylanases and glucosidases were up-regulated on 5 carbon sugars with respect to cellobiose. Chitinase and cellobiophosphorylases were the predominant CAZymes expressed on cellobiose. In addition to growth on xylan, the simultaneous consumption of two important lignocellulose constituents, cellobiose and xylose was also demonstrated.ConclusionThere are little changes in core-metabolic pathways under the different carbon sources compared. The most significant differences were found to be associated with the CAZymes, as well as specific up regulation of some key components of the pentose phosphate pathway in the presence of xylose and xylan. This study has enhanced our understanding of the physiology and metabolism of C. termitidis, and provides a foundation for future studies on metabolic engineering to optimize biofuel production from natural biomass.

Towards further defining the proteome of mouse saliva

BackgroundKnowledge of the mouse salivary proteome is not well documented and as a result, very limited. Currently, several salivary proteins remain unidentified and for some others, their function yet to be determined. The goal of the present study is to utilize mass spectrometry analysis to widen our knowledge of mouse salivary proteins, and through extensive database searches, provide further insight into the array of proteins that can be found in saliva. A comprehensive mouse salivary proteome will also facilitate the development of mouse models to study specific biomarkers of many human diseases.ResultsIndividual saliva samples were collected from male and female mice, and later pooled according to sex. Two pools of saliva from female mice (2 samples/pool) and 2 pools of saliva from male mice were used for analysis utilizing high performance liquid chromatograph mass spectrometry (nano-RPLC-MS/MS). The resulting datasets identified 345 proteins: 174 proteins were represented in saliva obtained from both sexes, as well as 82 others that were more female specific and 89 that were more male specific. Of these sex linked proteins, twelve were identified as exclusively sex-limited; 10 unique to males and 2 unique to females. Functional analysis of the 345 proteins identified 128 proteins with catalytic activity characteristics; indicative of proteins involved in digestion, and 35 proteins associated with stress response, host defense, and wound healing functions. Submission of the list of 345 proteins to the BioMart data mining tool in the Ensembl database further allowed us to identify a total of 283 orthologous human genes, of which, 131 proteins were recently reported to be present in the human salivary proteome.ConclusionsThe present study is the most comprehensive list to date of the proteins that constitute the mouse salivary proteome. The data presented can serve as a useful resource for identifying potentially useful biomarkers of human health and disease.

Unifying Expression Scale for Peptide Hydrophobicity in Proteomic Reversed Phase High-Pressure Liquid Chromatography Experiments

As an initial step in our efforts to unify the expression of peptide retention times in proteomic liquid chromatography-mass spectrometry (LC-MS) experiments, we aligned the chromatographic properties of a number of peptide retention standards against a collection of peptides commonly observed in proteomic experiments. The standard peptide mixtures and tryptic digests of samples of different origins were separated under the identical chromatographic condition most commonly employed in proteomics: 100 Å C18 sorbent with 0.1% formic acid as an ion-pairing modifier. Following our original approach (Krokhin, O. V.; Spicer, V. Anal. Chem. 2009, 81, 9522-9530) the retention characteristics of these standards and collection of tryptic peptides were mapped into hydrophobicity index (HI) or acetonitrile percentage units. This scale allows for direct visualization of the chromatographic outcome of LC-MS acquisitions, monitors the performance of the gradient LC system, and simplifies method development and interlaboratory data alignment. Wide adoption of this approach would significantly aid understanding the basic principles of gradient peptide RP-HPLC and solidify our collective efforts in acquiring confident peptide retention libraries, a key component in the development of targeted proteomic approaches.