Vic Spicer

Peptide Retention Standards and Hydrophobicity Indexes in Reversed-Phase High-Performance Liquid Chromatography of Peptides

The growing utility of peptide retention prediction in proteomics would benefit from the development of a universal peptide retention standard for better alignment of chromatographic data obtained using various liquid chromatography (LC) platforms. We describe a six-peptide mixture designed for this purpose; its members cover a wide range of hydrophobicity for the most popular modes of reversed-phase peptide high-performance liquid chromatography (HPLC): C18 sorbents with trifluoroacetic/formic acid as the ion-pairing modifier and separations at pH 10. We propose a hydrophobicity index (HI) describing the concentration of organic solvent (typically acetonitrile) that yields a retention factor of 10 under isocratic elution conditions for any peptide. This measure is a fundamental characteristic of peptide-sorbent interaction, depending only on the type of sorbent and the ion-pairing modifier used. Spiking a sample with a standard peptide mixture provides a measurement of the HI values of all detected species during gradient separation. In addition to alignment of data and calibration of chromatographic runs when peptide retention prediction protocols are used, these values obtained from proteomics experiments can be utilized directly in method development for large-scale preparative separations.

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.

Predicting Retention Time Shifts Associated with Variation of the Gradient Slope in Peptide RP-HPLC

We have developed a sequence-specific model for predicting slopes (S) in the fundamental equation of linear solvent strength theory for the reversed-phase HPLC separation of tryptic peptides detected in a typical bottom-up-proteomics experiment. These slopes control the variation in the separation selectivity observed when the physical parameters of chromatographic separation, such as gradient slope, flow rate, and column size are altered. For example, with the use of an arbitrarily chosen set of tryptic peptides with a 4-times difference in the gradient slope between two experiments, the R(2)-value of correlation between the observed retention times of identical species decreases to ~0.993-0.996 (compared to a theoretical value of ~1.00). The observed retention time shifts associated with variations of the gradient slope can be predicted a priori using the approach described here. The proposed model is based on our findings for a set of synthetic species (Vu, H.; Spicer, V.; Gotfrid, A.; Krokhin, O. V. J. Chromatogr., A, 2010, 1217, 489-497), which postulate that slopes S can be predicted taking into account simultaneously peptide length, charge, and hydrophobicity. Here we extend this approach using an extensive set of real tryptic peptides. We developed the procedure to accurately measure S-values in nano-RP HPLC MS experiments and introduced sequence-specific corrections for a more accurate prediction of the slopes S. A correlation of ~0.95 R(2)-value between the predicted and experimental S-values was demonstrated. Predicting S-values and calculating the expected retention time shifts when the physical parameters of separation like gradient slope are altered will facilitate a more accurate application of peptide retention prediction protocols, aid in the transfer of scheduled MRM (SRM) procedures between LC systems, and increase the efficiency of interlaboratory data collection and comparison.

Genomic Evaluation of Thermoanaerobacter spp. for the Construction of Designer Co-Cultures to Improve Lignocellulosic Biofuel Production

The microbial production of ethanol from lignocellulosic biomass is a multi-component process that involves biomass hydrolysis, carbohydrate transport and utilization, and finally, the production of ethanol. Strains of the genus Thermoanaerobacter have been studied for decades due to their innate abilities to produce comparatively high ethanol yields from hemicellulose constituent sugars. However, their inability to hydrolyze cellulose, limits their usefulness in lignocellulosic biofuel production. As such, co-culturing Thermoanaerobacter spp. with cellulolytic organisms is a plausible approach to improving lignocellulose conversion efficiencies and yields of biofuels. To evaluate native lignocellulosic ethanol production capacities relative to competing fermentative end-products, comparative genomic analysis of 11 sequenced Thermoanaerobacter strains, including a de novo genome, Thermoanaerobacter thermohydrosulfuricus WC1, was conducted. Analysis was specifically focused on the genomic potential for each strain to address all aspects of ethanol production mentioned through a consolidated bioprocessing approach. Whole genome functional annotation analysis identified three distinct clades within the genus. The genomes of Clade 1 strains encode the fewest extracellular carbohydrate active enzymes and also show the least diversity in terms of lignocellulose relevant carbohydrate utilization pathways. However, these same strains reportedly are capable of directing a higher proportion of their total carbon flux towards ethanol, rather than non-biofuel end-products, than other Thermoanaerobacter strains. Strains in Clade 2 show the greatest diversity in terms of lignocellulose hydrolysis and utilization, but proportionately produce more non-ethanol end-products than Clade 1 strains. Strains in Clade 3, in which T. thermohydrosulfuricus WC1 is included, show mid-range potential for lignocellulose hydrolysis and utilization, but also exhibit extensive divergence from both Clade 1 and Clade 2 strains in terms of cellular energetics. The potential implications regarding strain selection and suitability for industrial ethanol production through a consolidated bioprocessing co-culturing approach are examined throughout the manuscript.

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.

Thermoanaerobacter thermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates

ABSTRACT 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.

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.

Mass spectrometric study of N‐glycans from serum of woodchucks with liver cancer

Woodchucks have been a preferred lab animal model of chronic hepatitis B viral infection. The model recapitulates the disease progression of HBV infection to hepatocellular carcinoma (HCC) and has documented similarities in protein glycosylation with human HCC. This study examined N-glycans in serum of animals with(out) HCC. Oligosaccharides were released enzymatically using PNGaseF from total serum or from serum partially fractionated by extraction. Two different extraction procedures - reversed-phase high-performance liquid chromatography (RP-HPLC) and solid-phase extraction (SPE) on a cation-exchange/reversed-phase STRATA-XC cartridge - were used with the purpose of confirming glycosylation profiles. Oligosaccharides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) after derivatization with phenylhydrazine and/or permethylation. Characteristic fragment ions produced under MS/MS conditions allowed discrimination between isomeric structures of oligosaccharides, including those sialylated with two types of acidic residues. The complementary methods allowed structural characterization of oligosaccharides from various N-glycan classes. Furthermore, to validate results, glycosylation profiles of woodchuck sera were compared to glycans obtained from mouse serum on the same conditions. In summary, we have identified 40 N-glycan structures in the serum of woodchucks and some types of oligosaccharide structures appeared to increase in HCC samples following protease digest. The study provides improved tools for the characterization of N-glycans from total serum in the progression of liver disease.

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.

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 (R2 ∼ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R2 ∼ 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.