Analysis of the Yarrowia lipolytica proteome reveals subtle variations in expression levels between lipogenic and non-lipogenic conditions.

Oleaginous yeasts have the ability to store greater than 20% of their mass as neutral lipids, in the form of triacylglycerides. The ATP citrate lyase is thought to play a key role in triacylglyceride synthesis, but the relationship between expression levels of this and other related enzymes is not well understood in the role of total lipid accumulation conferring the oleaginous phenotype. We conducted comparative proteomic analyses with the oleaginous yeast, Yarrowia lipolytica, grown in either nitrogen-sufficient rich media or nitrogen-limited minimal media. Total proteins extracted from cells collected during logarithmic and late stationary growth phases were analyzed by 1D liquid chromatography, followed by mass spectroscopy. The ATP citrate lyase enzyme was expressed at similar concentrations in both conditions, in both logarithmic and stationary phase, but many upstream and downstream enzymes showed drastically different expression levels. In non-lipogenic conditions, several pyruvate enzymes were expressed at higher concentration. These enzymes, especially the pyruvate decarboxylase and pyruvate dehydrogenase, may be regulating carbon flux away from central metabolism and reducing the amount of citrate being produced in the mitochondria. While crucial for the oleaginous phenotype, the constitutively expressed ATP citrate lyase appears to cleave citrate in response to carbon flux upstream from other enzymes creating the oleaginous phenotype.

Peptide separation selectivity in proteomics LC-MS experiments: Comparison of formic and mixed formic/heptafluorobutyric acids ion-pairing modifiers.

Separation selectivity and detection sensitivity of reversed-phase high-performance liquid chromatography with tandem mass spectrometry analyses were compared for formic (0.1%) and formic/heptafluorobutyric (0.1%/0.005%) acid based eluents using a proteomic data set of ∼12 000 paired peptides. The addition of a small amount of hydrophobic heptafluorobutyric acid ion-pairing modifier increased peptide retention by up to 10% acetonitrile depending on peptide charge, size, and hydrophobicity. Retention increase was greatest for peptides that were short, highly charged, and hydrophilic. There was an ∼3.75-fold reduction in MS signal observed across the whole population of peptides following the addition of heptafluorobutyric acid. This resulted in ∼36% and ∼21% reduction of detected proteins and unique peptides for the whole cell lysate digests, respectively. We also confirmed that the separation selectivity of the formic/heptafluorobutyric acid system was very similar to the commonly used conditions of 0.1% trifluoroacetic acid, and developed a new version of the Sequence-Specific Retention calculator model for the formic/heptafluorobutyric acid system showing the same ∼0.98 R2 -value accuracy as the Sequence-Specific Retention calculator formic acid model. In silico simulation of peptide distribution in separation space showed that the addition of 0.005% heptafluorobutyric acid to the 0.1% formic acid system increased potential proteome coverage by ∼11% of detectable species (tryptic peptides ≥ four amino acids).

Retention Time Prediction for Glycopeptides in Reversed-Phase Chromatography for Glycoproteomic Applications.

The sequence-specific retention calculator algorithm (SSRCalc) [ Krokhin , O. V. Anal. Chem. 2006 , 78 , 7785 ] was adapted for the prediction of retention times of N-glycopeptides separated by reversed-phase high performance liquid chromatography (RPLC). The retention time shifts (dHI = HIglyco - HIdeglyco, where HI is the hydrophobicity index, measured in percent acetonitrile units) used for modeling were measured for 602 glycopeptides versus 123 of their deglycosylated analogues. Our method used a tryptic digest of 12 purified glycoproteins, glycopeptide enrichment, deglycosylation with PNGaseF, and RPLC-MS/MS analysis of combined (deglycosylated and intact) peptide mixtures. On average, glycosylation yields a 0.79% acetonitrile unit decrease in retention, compared with the hydrophobicity indices of their deglycosylated analogues. These values, however, are drastically different for asialo (-1.37% acetonitrile units), monosialylated (-0.47% acetonitrile units), disialylated (+0.61% acetonitrile units), and trisialylated (+1.94% acetonitrile units) glycans. Peptide retention time shifts upon glycosylation (dHI) vary depending on the number of monosaccharide units, the presence or absence of sialic acid, peptide hydrophobicity, and the number of position-dependent features. These features are mostly driven by competing effects of acidic residues (aspartic acid and sialic acid) on ion-pair formation and by nearest-neighbor effects of hydrophilic glycans. The accuracy of the modified prediction model for glycopeptides approaches that of the prediction for nonmodified species (R2 = 0.97 vs 0.98). However, retention time prediction based on the experimental retention values of deglycosylated analogues (HIglyco = HIdeglyco + dHI, R2 = 0.995) is much more accurate, thus providing a solid support for glycopeptide identification in complex samples based on mass and retention time.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line.

Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide data set is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Phosphopeptides migrate significantly slower than corresponding unphosphopeptides under acidic conditions of CZE separations and in a normal polarity. According to our modeling data, phosphorylation decreases peptide's charge roughly by one charge unit, resulting in dramatic decrease in electrophoretic mobility. Preliminary investigations demonstrate that electrophoretic mobility of phosphopeptides containing one phosphoryl group can be predicted with the same accuracy as for nonmodified peptides ( R2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.

A metabolic and genomic assessment of sugar fermentation profiles of the thermophilic Thermotogales, Fervidobacterium pennivorans.

A metabolic, genomic and proteomic assessment of Fervidobacterium pennivorans strains was undertaken to clarify the metabolic and genetic capabilities of this Thermotogales species. The type strain Ven5 originally isolated from a hot mud spa in Italy, and a newly isolated strain (DYC) from a hot spring at Ngatamariki, New Zealand, were compared for metabolic and genomic differences. The fermentation profiles of both strains on cellobiose generated similar major end products (acetate, alanine, glutamate, H2, and CO2). The vast majority of end products produced were redox neutral, and carbon balances were in the range of 95-115%. Each strain showed distinct fermentation profiles on sugar substrates. The genome of strain DYC was sequenced and shown to have high sequence similarity and synteny with F. pennivorans Ven5 genome, suggesting they are the same species. The unique genome regions in Ven5, corresponded to genes involved in the Entner-Doudoroff pathway confirming our observation of DYC's inability to utilize gluconate. Genome analysis was able to elucidate pathways involved in production of the observed end-products with the exception of alanine and glutamate synthesis which were resolved with less clarity due to poor sequence identity and missing critical enzymes within the pathway, respectively.

Peptide Retention Time Prediction in Hydrophilic Interaction Liquid Chromatography: Data Collection Methods and Features of Additive and Sequence-Specific Models.

The development of a peptide retention prediction model for hydrophilic interaction liquid chromatography (XBridge Amide column) is described for a collection of ∼40 000 tryptic peptides. Off-line 2D LC-MS/MS analysis (HILIC-RPLC) of S. cerevisiae whole cell lysate has been used to acquire retention information for a HILIC separation. The large size of the optimization data set (more than 2 orders of magnitude compared to previous reports) permits the accurate assignment of hydrophilic retention coefficients of individual amino acids, establishing both the effects of amino acid position relative to peptide termini and the influence of peptide secondary structure in HILIC. The accuracy of a simple additive model with peptide length correction (R2 value of ∼0.96) was found to be much higher compared to an algorithm of similar complexity applied to RPLC (∼0.91). This indicates significantly smaller influence of peptide secondary structure and interactions with counterions in HILIC. Nevertheless, sequence-specific features were found. Helical peptides that tend to retain stronger than predicted in RPLC exhibit negative prediction errors using an additive HILIC model. N-cap helix stabilizing motifs, which increase retention of amphipathic helical peptides in RP, reduce peptide retention in HILIC independently of peptide amphipathicity. Peptides carrying multiple Pro and Gly (residues with lowest helical propensity) retain stronger than predicted. We conclude that involvement of the peptide backbone's carbonyl and amide groups in hydrogen-bond stabilization of helical structures is a major factor, which determines sequence-dependent behavior of peptides in HILIC. The incorporation of observed sequence dependent features into our Sequence-Specific Retention Calculator HILIC model resulted in 0.98 R2 value correlation, significantly exceeding the predictive performance of all RP and HILIC models developed for complex mixtures of tryptic peptides.

Predicting Electrophoretic Mobility of Tryptic Peptides for High-Throughput CZE-MS Analysis.

A multiparametric sequence-specific model for predicting peptide electrophoretic mobility has been developed using large-scale bottom-up proteomic CE-MS data (5% (∼0.8M) acetic acid as background electrolyte). Peptide charge (Z) and size (molecular mass, M) are the two major factors determining electrophoretic mobility, in complete agreement with previous studies. The extended size of the data set (>4000 peptides) permits access to many sequence-specific factors that impact peptide mobility. The presence of acidic residues Asp and Glu near the peptide N-terminus is by far the most prominent among them. The induction effect of the side chain of N-terminal Asp reduces the basicity of the N-terminal amino group and, as hence, its charge, by ∼0.27 units, lowering mobility. The correlation of the model (R2 ∼ 0.995) indicates that the peptide separation process in CZE is relatively simple and can be predicted to a much higher precision than current RP-HPLC models. Similar to RP-HPLC prediction studies, we anticipate future developments that introduce peptide migration standards, collect larger data sets for modeling through the alignment of multiple CZE-MS acquisitions, and study of the behavior of peptides carrying post-translational modifications. The increased size of data sets will also permit investigation of the fine-scale effects of peptide secondary structure on peptide mobility. We observed that peptides with higher helical propensity tend to have higher than predicted electrophoretic mobility; the incorporation of these features into CZE migration models will require significantly larger data sets.

Generation of accurate peptide retention data for targeted and data independent quantitative LC-MS analysis: Chromatographic lessons in proteomics.

The emergence of data-independent quantitative LC-MS/MS analysis protocols further highlights the importance of high-quality reproducible chromatographic procedures. Knowing, controlling and being able to predict the effect of multiple factors that alter peptide RP-HPLC separation selectivity is critical for successful data collection for the construction of ion libraries. Proteomic researchers have often regarded RP-HPLC as a "black box", while vast amount of research on peptide separation is readily available. In addition to obvious parameters, such as the type of ion-pairing modifier, stationary phase and column temperature, we describe the "mysterious" effects of gradient slope, column size and flow rate on peptide separation selectivity. Retention time variations due to these parameters are governed by the linear solvent strength (LSS) theory on a peptide level by the value of its slope S in the basic LSS equation-a parameter that can be accurately predicted. Thus, the application of shallower gradients, higher flow rates, or smaller columns will each increases the relative retention of peptides with higher S-values (long species with multiple positively charged groups). Simultaneous changes to these parameters that each drive shifts in separation selectivity in the same direction should be avoided. The unification of terminology represents another pressing issue in this field of applied proteomics that should be addressed to facilitate further progress.

3D HPLC-MS with Reversed-Phase Separation Functionality in All Three Dimensions for Large-Scale Bottom-Up Proteomics and Peptide Retention Data Collection.

The growing complexity of proteomics samples and the desire for deeper analysis drive the development of both better MS instrument and advanced multidimensional separation schemes. We applied 1D, 2D, and 3D LC-MS/MS separation protocols (all of reversed-phase C18 functionality) to a tryptic digest of whole Jurkat cell lysate to estimate the depth of proteome coverage and to collect high-quality peptide retention information. We varied pH of the eluent and hydrophobicity of ion-pairing modifier to achieve good separation orthogonality (utilization of MS instrument time). All separation modes employed identical LC settings with formic-acid-based eluents in the last dimension. The 2D protocol used a high pH-low pH scheme with 21 concatenated fractions. In the 3D protocol, six concatenated fractions from the first dimension (C18, heptafluorobutyric acid) were analyzed using the identical 2D LC-MS procedure. This approach permitted a detailed evaluation of the analysis output consuming 21× and 126× the analysis time and sample load compared to 1D. Acquisition over 189 h of instrument time in 3D mode resulted in the identification of ∼14 000 proteins and ∼250 000 unique peptides. We estimated the dynamic range via peak intensity at the MS(2) level as approximately 10(4.2), 10(5.6), and 10(6.2) for the 1D, 2D, and 3D protocols, respectively. The uniform distribution of the number of acquired MS/MS, protein, and peptide identifications across all 126 fractions and through the chromatographic time scale in the last LC-MS stage indicates good separation orthogonality. The protocol is scalable and is amenable to the use of peptide retention prediction in all dimensions. All these features make it a very good candidate for large-scale bottom-up proteomic runs, which target both protein identification as well as the collection of peptide retention data sets for targeted quantitative applications.

Whole cell, label free protein quantitation with data independent acquisition: quantitation at the MS2 level.

Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R(2) ∼ 0.930), indicating that this approach may have increased viability compared to recent applications in label free protein quantitation. SWATH based quantitation was able to consistently detect differences in relative protein quantity and it provided coverage for a number of proteins that were missed in some samples by DDA analysis.

Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

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.

Thermoanaerobacter thermohydrosulfuricus WC1 shows protein complement stability during fermentation of key lignocellulose-derived substrates.

Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using "omic"-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain, Thermoanaerobacter thermohydrosulfuricus WC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species "omic" comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverse Thermoanaerobacter spp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.

The requirement for the LysR-type regulator PtrA for Pseudomonas chlororaphis PA23 biocontrol revealed through proteomic and phenotypic analysis.

BACKGROUND: Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the antibiotics phenazine and pyrrolnitrin together with other metabolites believed to contribute to biocontrol. A mutant no longer capable of inhibiting fungal growth was identified harboring a transposon insertion in a gene encoding a LysR-type transcriptional regulator (LTTR), designated ptrA (Pseudomonas transcriptional regulator). Isobaric tag for relative and absolute quantitation (iTRAQ) based protein analysis was used to reveal changes in protein expression patterns in the ptrA mutant compared to the PA23 wild type. RESULTS: Relative abundance profiles showed 59 differentially-expressed proteins in the ptrA mutant, which could be classified into 16 clusters of orthologous groups (COGs) based on their predicted functions. The largest COG category was the unknown function group, suggesting that many yet-to-be identified proteins are involved in the loss of fungal activity. In the secondary metabolite biosynthesis, transport and catabolism COG, seven proteins associated with phenazine biosynthesis and chitinase production were downregulated in the mutant. Phenotypic assays confirmed the loss of phenazines and chitinase activity. Upregulated proteins included a lipoprotein involved in iron transport, a flagellin and hook-associated protein and four proteins categorized into the translation, ribosome structure and biogenesis COG. Phenotypic analysis revealed that the mutant exhibited increased siderophore production and flagellar motility and an altered growth profile, supporting the proteomic findings. CONCLUSION: PtrA is a novel LTTR that is essential for PA23 fungal antagonism. Differential protein expression was observed across 16 COG categories suggesting PtrA is functioning as a global transcriptional regulator. Changes in protein expression were confirmed by phenotypic assays that showed reduced phenazine and chitinase expression, elevated flagellar motility and siderophore production, as well as early entrance into log phase growth.

Retention time prediction for post-translationally modified peptides: Ser, Thr, Tyr-phosphorylation.

The development of a peptide retention prediction model for reversed-phase chromatography applications in proteomics is reported for peptides carrying phosphorylated Ser, Thr and Tyr-residues. The major retention features have been assessed using a collection of over 10,000 phosphorylated/non-phosphorylated peptide pairs identified in a series 1D and 2D LC-MS/MS acquisitions using formic acid as ion pairing modifier. Single modification event on average results in increased peptide retention for phosphorylation of Ser (+ 1.46), Thr (+1.33), Tyr (+0.93% acetonitrile, ACN) on gradient elution scale for Luna C18(2) stationary phase. We established several composition and sequence specific features, which drive deviations from these average values. Thus, single phosphorylation of serine results in retention shifts ranging from -2.4 to 5.5% ACN depending on position of the residue, nature of nearest neighbour residues, peptide length, hydrophobicity and pI value, and its propensity to form amphipathic helical structures. We established that the altered ion-pairing environment upon phosphorylation is detrimental for this variability. Hydrophobicity of ion-pairing modifier directly informs the magnitude of expected shifts: (most hydrophilic) 0.5 % acetic acid (larger positive shift upon phosphorylation) > 0.1 % formic acid (positive) > 0.1 % trifluoroacetic (negative) > 0.1 % heptafluorobutyric acid (larger negative shift). The effect of phosphorylation has been also evaluated for several separation conditions used in the first dimension of 2D LC applications: high pH reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), strong cation- and strong anion exchange separations.

Alterations in glycopeptides associated with herceptin treatment of human breast carcinoma mcf-7 and T-lymphoblastoid cells.

The therapeutic humanized monoclonal antibody IgG1 known as Herceptin® has shown remarkable antitumor effects. Although this type of therapy has increased the cancer-free survival of patients, not all tumors respond to this treatment and cancers often develop resistance to the antibody. Despite the fact that Herceptin function has been extensively studied, the precise mechanism underlying its antitumor activity still remains incompletely defined. We previously demonstrated on human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells that monoclonal antibody in combination with Lipoplex consisting of Lipofectamine mixed with plasmid DNA showed a more profound effect on cancer cell viability than antibody alone. The analyses of N-glycans isolated from cancer cells showed dramatic differences in profiles when cells were exposed to Herceptin. Moreover, the investigation of glycosylated peptides from the same cancer cell models after treatment revealed further alterations in the post-translational modifications. Tandem mass spectra obtained from the samples treated confirmed the presence of a series of glycopeptides bearing characteristic oligosaccharides as described in IgG1. However some of them differed by mass differences that corresponded to peptide backbones not described previously and more of them were detected from Herceptin treated samples than from cells transfected with Heceptin/Lipoplex. The results indicate that the presence of Lipoplex prevents antibody transformation and elongates its proper function. The better understanding of the multipart changes described in the glycoconjugates could provide new insights into the mechanism by which antibody induces regression in cancers.

Deep learning from harmonized peptide libraries enables retention time prediction of diverse post translational modifications.

In proteomics experiments, peptide retention time (RT) is an orthogonal property to fragmentation when assessing detection confidence. Advances in deep learning enable accurate RT prediction for any peptide from sequence alone, including those yet to be experimentally observed. Here we present Chronologer, an open-source software tool for rapid and accurate peptide RT prediction. Using new approaches to harmonize and false-discovery correct across independently collected datasets, Chronologer is built on a massive database with >2.2 million peptides including 10 common post-translational modification (PTM) types. By linking knowledge learned across diverse peptide chemistries, Chronologer predicts RTs with less than two-thirds the error of other deep learning tools. We show how RT for rare PTMs, such as OGlcNAc, can be learned with high accuracy using as few as 10-100 example peptides in newly harmonized datasets. This iteratively updatable workflow enables Chronologer to comprehensively predict RTs for PTM-marked peptides across entire proteomes.

Information-dependent LC-MS/MS acquisition with exclusion lists potentially generated on-the-fly: case study using a whole cell digest of Clostridium thermocellum.

We have developed a real-time graphic-processor-unit-based search engine capable of high-quality peptide identifications in <500 µs per spectrum. The steps of peptide/protein identification, in-silico prediction of all possible tryptic peptides from these proteins, and the prediction of their expected retention times and m/z values take less than 5 s per cycle over ∼3000 MS/MS spectra. This lays the foundation for information-dependent acquisition with exclusion lists generated on-the-fly, as the instrument continues to acquire data. While a complete evaluation of the dynamic exclusion system requires the participation from instrument vendors, we conducted a series of model experiments using a whole cell tryptic digestion of the bacterium Clostridium thermocellum. We ran a series of five iterative LC-MS/MS runs, adding a new exclusion list at each of four chromatographic "tripping points" - the elution times of the four standard peptides spiked into the sample. Retention times of these standard peptides were also used for real-time "chromatographic calibration." The dynamic exclusion approach gave a ≈ 5% increase in confident protein identification (for typical 2 h LC-MS/MS run), and reduced the average number of identified peptides per protein from 4.7 to 2.9. Its application to a two-times shorter gradient gave a ≈ 17% increase in proteins identified. Further improvements are possible for instruments with better mass accuracy, by employing a more accurate retention prediction algorithm and by developing better understanding of the possible chemical modifications and fragmentations produced during electrospray ionization.

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

BACKGROUND: Clostridium 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. RESULTS: Relative 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. CONCLUSIONS: Relative 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.

Chromatographic behaviour of peptides modified with amine-reacting tags for relative protein quantitation in proteomic applications.

Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts ΔHI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs.