Thermodynamic Integration Networks and Their Application to Charge Transfer Reactions within the AauDyPI Fungal Peroxidase.

We present a computer simulation study of the thermodynamics and kinetics of charge transfer reactions within the fungal peroxidase AauDyPI from Auricularia auriculae-judae. Driving forces and reorganization energies are obtained from a thermodynamic integration scheme based upon molecular dynamics simulations. To enhance the numerical accuracy, the free energies are analyzed within a least-squares scheme of a closely knit thermodynamic network.

Oxidation and nitration of mononitrophenols by a DyP-type peroxidase.

Substantial conversion of nitrophenols, typical high-redox potential phenolic substrates, by heme peroxidases has only been reported for lignin peroxidase (LiP) so far. But also a dye-decolorizing peroxidase of Auricularia auricula-judae (AauDyP) was found to be capable of acting on (i) ortho-nitrophenol (oNP), (ii) meta-nitrophenol (mNP) and (iii) para-nitrophenol (pNP). The pH dependency for pNP oxidation showed an optimum at pH 4.

The toolbox of Auricularia auricula-judae dye-decolorizing peroxidase - Identification of three new potential substrate-interaction sites.

Dye-decolorizing peroxidases (DyPs) such as AauDyPI from the fungus Auricularia auricula-judae are able to oxidize substrates of different kinds and sizes. A crystal structure of an AauDyPI-imidazole complex gives insight into the binding patterns of organic molecules within the heme cavity of a DyP. Several small N-containing heterocyclic aromatics are shown to bind in the AauDyPI heme cavity, hinting to susceptibility of DyPs to azole-based inhibitors similar to cytochromes P450.

Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits.

Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.

Radical formation on a conserved tyrosine residue is crucial for DyP activity.

Dye-decolorizing peroxidases (DyPs) are able to cleave bulky anthraquinone dyes. The recently published crystal structure of AauDyPI reveals that a direct oxidation in the distal heme cavity can be excluded for most DyP substrates. It is shown that a surface-exposed tyrosine residue acts as a substrate interaction site for bulky substrates.

First crystal structure of a fungal high-redox potential dye-decolorizing peroxidase: substrate interaction sites and long-range electron transfer.

Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. AauDyPI from Auricularia auricula-judae (fungi) was crystallized, and its crystal structure was determined at 2.

Identification of missing genes and enzymes for autotrophic carbon fixation in crenarchaeota.

Two autotrophic carbon fixation cycles have been identified in Crenarchaeota. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the Thermoproteales and Desulfurococcales. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic Sulfolobales; a similar cycle may operate in autotrophic aerobic marine Crenarchaeota.

Post-translational modifications of chicken myelin basic protein charge components.

Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris.

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.

Improved peptide elution time prediction for reversed-phase liquid chromatography-MS by incorporating peptide sequence information.

We describe an improved artificial neural network (ANN)-based method for predicting peptide retention times in reversed-phase liquid chromatography. In addition to the peptide amino acid composition, this study investigated several other peptide descriptors to improve the predictive capability, such as peptide length, sequence, hydrophobicity and hydrophobic moment, and nearest-neighbor amino acid, as well as peptide predicted structural configurations (i.e.

A proteomic view of Desulfovibrio vulgaris metabolism as determined by liquid chromatography coupled with tandem mass spectrometry.

Direct LC-MS/MS was used to examine the proteins extracted from exponential or stationary phase Desulfovibrio vulgaris cells that had been grown on a minimal medium containing either lactate or formate as the primary carbon source. Across all four growth conditions, 976 gene products were identified with high confidence, which is equal to approximately 28% of all predicted proteins in the D. vulgaris genome.

Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation.

A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level.

Quantitative proteome analysis of breast cancer cell lines using 18O-labeling and an accurate mass and time tag strategy.

Proteome comparison of cell lines derived from cancer and normal breast epithelium provide opportunities to identify differentially expressed proteins and pathways associated with specific phenotypes. We employed 16O/18O peptide labeling, FT-ICR MS, and an accurate mass and time (AMT) tag strategy to simultaneously compare the relative abundance of hundreds of proteins in non-cancer and cancer cell lines derived from breast tissue. A cell line reference panel allowed relative protein abundance comparisons among multiple cell lines and across multiple experiments.

Comparison of normal and breast cancer cell lines using proteome, genome, and interactome data.

Normal and cancer cell line proteomes were profiled using high throughput mass spectrometry techniques. Application of protein-level and peptide-level sample fractionation combined with LC-MS/MS analysis enabled identification of 2235 unmodified proteins representing a broad range of functional and compartmental classes. An iterative multistep search strategy was used to identify post-translational modifications, revealing several proteins that are preferentially modified in cancer cells.

Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurements.

The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through on-line electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.

Two-dimensional gas-phase separations coupled to mass spectrometry for analysis of complex mixtures.

Ion mobility spectrometry (IMS) has been explored for decades, and its versatility in separation and identification of gas-phase ions is well established. Recently, field asymmetric waveform IMS (FAIMS) has been gaining acceptance in similar applications. Coupled to mass spectrometry (MS), both IMS and FAIMS have shown the potential for broad utility in proteomics and other biological analyses.

Characterization of the human blood plasma proteome.

We describe methods for broad characterization of the human plasma proteome. The combination of stepwise immunoglobulin G (IgG) and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of > 94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (< 30 pg/mL to approximately 30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods.

Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry.

We describe methods for mass spectrometric identification of heme-containing peptides from c-type cytochromes that contain the CXXCH (X=any amino acid) sequence motif. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference for both 2+ and 3+ peptide charge states; both sequence and charge affect the extent of heme loss. Application to Shewanella oneidenis demonstrated the utility of this approach for identifying c-type heme-containing peptides from complex proteome samples.

Identification of shed proteins from Chinese hamster ovary cells: application of statistical confidence using human and mouse protein databases.

The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation to develop a fundamental understanding of the bystander response.

Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis: the human proteome.

Large-scale protein identifications from highly complex protein mixtures have recently been achieved using multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) and subsequent database searching with algorithms such as SEQUEST. Here, we describe a probability-based evaluation of false positive rates associated with peptide identifications from three different human proteome samples. Peptides from human plasma, human mammary epithelial cell (HMEC) lysate, and human hepatocyte (Huh)-7.

High-throughput comparative proteome analysis using a quantitative cysteinyl-peptide enrichment technology.

A new quantitative cysteinyl-peptide enrichment technology (QCET) was developed to achieve higher efficiency, greater dynamic range, and higher throughput in quantitative proteomics that use stable-isotope labeling techniques combined with high-resolution liquid chromatography (LC)-mass spectrometry (MS). This approach involves (18)O labeling of tryptic peptides, high-efficiency enrichment of cysteine-containing peptides, and confident protein identification and quantification using the accurate mass and time tag strategy. Proteome profiling of naïve and in vitro-differentiated human mammary epithelial cells using QCET resulted in the identification and quantification of 603 proteins in a single LC-Fourier transform ion cyclotron resonance MS analysis.

Application of peptide LC retention time information in a discriminant function for peptide identification by tandem mass spectrometry.

We describe the application of a peptide retention time reversed phase liquid chromatography (RPLC) prediction model previously reported (Petritis et al. Anal. Chem.

Multidimensional proteome analysis of human mammary epithelial cells.

Recent multidimensional liquid chromatography MS/MS studies have contributed to the identification of large numbers of expressed proteins for numerous species. The present study couples size exclusion chromatography of intact proteins with the separation of tryptically digested peptides using a combination of strong cation exchange and high resolution, reversed phase capillary chromatography to identify proteins extracted from human mammary epithelial cells (HMECs). In addition to conventional conservative criteria for protein identifications, the confidence levels were additionally increased through the use of peptide normalized elution times (NET) for the liquid chromatographic separation step.

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics.

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument.

Automated gain control and internal calibration with external ion accumulation capillary liquid chromatography-electrospray ionization Fourier transform ion cyclotron resonance.

When combined with capillary LC separations, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) has demonstrated capabilities for advanced characterization of proteomes based upon analyses of proteolytic digests. Incorporation of external (to the ICR cell) multipole devices with FTICR for ion selection and ion accumulation has enhanced the dynamic range, sensitivity, and duty cycle of measurements. However, the highly variable ion production rate from an LC separation can result in "overfilling" of the external trap during the elution of major peaks and result in m/z discrimination and fragmentation of peptide ions.