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Paul A Haynes

Publications and source records attributed to Paul A Haynes.

At least 19 recordsLinked to original sources
PubMed2025

Uncovering the antifungal potential of Cannabidiol and Cannabidivarin.

Fungal infections pose a major threat to human health with increasing incidence of antifungal resistance globally. Despite the need for novel antifungal drugs, few are currently in clinical development. Here we evaluate the antifungal activity of five phytocannabinoids against several clinically relevant fungal pathogens, with a focus on the priority pathogen Cryptococcus neoformans. Our results demonstrate that Cannabidiol (CBD), and particularly Cannabidivarin (CBDV), have broad activity against C. neoformans and other fungal pathogens, including dermatophytes that cause common tinea. We found that both CBD and CBDV acted in a fungicidal manner and prevented biofilm formation in C. neoformans. Phytocannabinoid treatment impeded factors important for virulence and antifungal resistance, including reduced capsule size and disruption of mature biofilms. Proteomics analysis revealed that the antifungal activity of CBD and CBDV was linked to destabilisation of the membrane, alterations in ergosterol biosynthesis, disruption of metabolic pathways, as well as selective involvement of mitochondrial-associated proteins. We next tested the ability of CBD to topically clear a C. neoformans fungal infection in vivo using the Galleria mellonella burn wound model, and we observed greatly improved survival in the CBD treated larvae. This study illustrates the potential of phytocannabinoids as antifungal treatments and opens up new routes towards development of novel antifungal drugs.

Antifungal Agents↗
PubMed2007

Two-dimensional differential in-gel electrophoresis (DIGE) of leaf and roots of Lycopersicon esculentum.

In this report we present a detailed protocol for the analysis of differential protein expression between two plant tissue samples. The protocol involves harvesting of leaves and roots from mature tomato plants, preparing protein extracts from the harvested tissues, fluorescent labeling of each sample prior to differential in-gel electrophoresis (DIGE), first- and second-dimension electrophoretic separations, and image analysis to visualize and quantify differential protein expression. This protocol is adaptable for use with a wide variety of plant materials and can be used to measure protein expression changes occurring in response to abiotic stress, biotic stress, genetic manipulation, selective breeding, and many other conditions. In addition to the detailed protocol, we also present the results of a representative experiment analyzing subtle changes in protein expression in the roots of tomato plants grown under control and salt-stress conditions.

Acetone↗
PubMed2007

Two-dimensional nanoflow liquid chromatography-tandem mass spectrometry of proteins extracted from rice leaves and roots.

In this chapter we present a detailed protocol for the large-scale identification of proteins present in rice leaf and root tissue samples using 2D liquid chromatography-tandem mass spectrometry of protein extracts. This is performed using biphasic (strong cation exchange/reversed phase) columns with integral electrospray emitters operating at nanoliter flow rates, a technique known by the acronym Mudpit (for multidimensional protein identification technique). The protocol involves harvesting of leaves and roots from rice plants, preparing protein extracts from the harvested tissues, preparing proteolytic digests of the extracted proteins, making a biphasic capillary column with an integral electrospray emitter, performing two-dimensional chromatographic separation of peptides with data-dependent tandem mass spectrometry, and the use of database searching of the acquired tandem mass spectra to identify peptides and proteins. This protocol is adaptable for use with a wide variety of plant materials and can be used to identify large numbers of proteins present in a specific tissue, organ, organelle, or other subcellular fraction. In addition to the detailed protocol, we also present the results of a representative experiment showing the identification of more than 1000 distinct proteins from rice leaf and root samples in two Mupdit experiments.

Acetone↗
PubMed2006

Protein expression profiling of Coccidioides posadasii by two-dimensional differential in-gel electrophoresis and evaluation of a newly recognized peroxisomal matrix protein as a recombinant vaccine candidate.

Coccidioides posadasii and Coccidioides immitis are dimorphic, soil-dwelling pathogenic ascomycetes endemic to the southwestern United States. Infection can result from inhalation of a very few arthroconidia, but following natural infection, long-lived immunity is the norm. Previous work in the field has shown that spherule-derived vaccines afford more protection than those from mycelia. We have used two-dimensional differential in-gel electrophoresis coupled with nano-high-performance liquid chromatography-tandem mass spectrometry to directly assess both absolute abundance and differential expression of proteins in the spherule and the mycelial phases of C. posadasii with the intent to identify potential vaccine candidates. Peptides derived from 40 protein spots were analyzed and a probable identity was assigned to each. One spherule-abundant protein, identified as Pmp1, showed homology to allergens from Aspergillus fumigatus and other fungi, all of which exhibit similarity to yeast thiol peroxidases. Recombinant Pmp1 was reactive with serum from individuals with both acute and protracted disease, and evoked protection in two murine models of infection with C. posadasii. These results demonstrate the utility of proteomic analysis as a point of discovery for protective antigens for possible inclusion in a vaccine candidate to prevent coccidioidomycosis.

Animals↗
PubMed2006

The wildcat toolbox: a set of perl script utilities for use in peptide mass spectral database searching and proteomics experiments.

We describe in this communication a set of functional perl script utilities for use in peptide mass spectral database searching and proteomics experiments, known as the Wildcat Toolbox. These are all freely available for download from our laboratory Web site (http://proteomics.arizona.edu/toolbox.html) as a combined zip file, and can also be accessed via the Proteome Commons Web site (www.proteomecommons.org) in the tools section. We make them available to other potential users in the spirit of open source software development; we do not have the resources to provide any significant technical support for them, but we hope users will share both bugs and improvements with the community at large.

Algorithms↗
PubMed2006

Verification of single-peptide protein identifications by the application of complementary database search algorithms.

Data produced from the MudPIT analysis of yeast (S. cerevisiae) and rice (O. sativa) were used to develop a technique to validate single-peptide protein identifications using complementary database search algorithms. This results in a considerable reduction of overall false-positive rates for protein identifications; the overall false discovery rates in yeast are reduced from near 25% to less than 1%, and the false discovery rate of yeast single-peptide protein identifications becomes negligible. This technique can be employed by laboratories utilizing a SEQUEST-based proteomic analysis platform, incorporating the XTandem algorithm as a complementary tool for verification of single-peptide protein identifications. We have achieved this using open-source software, including several data-manipulation software tools developed in our laboratory, which are freely available to download.

Algorithms↗
PubMed2005

Identification of proteins from a cell wall fraction of the diatom Thalassiosira pseudonana: insights into silica structure formation.

Diatoms are unicellular eucaryotic algae with cell walls containing silica, intricately and ornately structured on the nanometer scale. Overall silica structure is formed by expansion and molding of the membrane-bound silica deposition vesicle. Although molecular details of silica polymerization are being clarified, we have limited insight into molecular components of the silica deposition vesicle, particularly of membrane-associated proteins that may be involved in structure formation. To identify such proteins, we refined existing procedures to isolate an enriched cell wall fraction from the diatom Thalassiosira pseudonana, the first diatom with a sequenced genome. We applied tandem mass spectrometric analysis to this fraction, identifying 31 proteins for further evaluation. mRNA levels for genes encoding these proteins were monitored during synchronized progression through the cell cycle and compared with two previously identified silaffin genes (involved in silica polymerization) having distinct mRNA patterns that served as markers for cell wall formation. Of the 31 proteins identified, 10 had mRNA patterns that correlated with the silaffins, 13 had patterns that did not, and seven had patterns that correlated but also showed additional features. The possible involvements of these proteins in cell wall synthesis are discussed. In particular, glutamate acetyltransferase was identified, prompting an analysis of mRNA patterns for other genes in the polyamine biosynthesis pathway and identification of those induced during cell wall synthesis. Application of a specific enzymatic inhibitor for ornithine decarboxylase resulted in dramatic alteration of silica structure, confirming the involvement of polyamines and demonstrating that manipulation of proteins involved in cell wall synthesis can alter structure. To our knowledge, this is the first proteomic analysis of a diatom, and furthermore we identified new candidate genes involved in structure formation and directly demonstrated the involvement of one enzyme (and its gene) in the structure formation process.

Acetyltransferases↗
PubMed2005

Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals.

We have conducted a proteomic analysis of the 80S cytosolic ribosome from the eukaryotic green alga Chlamydomonas reinhardtii, and accompany this with a cryo-electron microscopy structure of the ribosome. Proteins homologous to all but one rat 40S subunit protein, including a homolog of RACK1, and all but three rat 60S subunit proteins were identified as components of the C. reinhardtii ribosome. Expressed Sequence Tag (EST) evidence and annotation of the completed C. reinhardtii genome identified genes for each of the four proteins not identified by proteomic analysis, showing that algae potentially have a complete set of orthologs to mammalian 80S ribosomal proteins. Presented at 25A, the algal 80S ribosome is very similar in structure to the yeast 80S ribosome, with only minor distinguishable differences. These data show that, although separated by billions of years of evolution, cytosolic ribosomes from photosynthetic organisms are highly conserved with their yeast and animal counterparts.

Algal Proteins↗
PubMed2005

Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing.

We have investigated the use of a variety of different techniques to identify as many proteins as possible in a yeast lysate, with the aim of investigating the overlap and complementarity of data from different approaches. A standard lysate was prepared from log phase yeast (Saccharomyces cerevisiae). This was then subjected to analysis via five different approaches aimed at identifying as many proteins as possible using an ion trap mass spectrometer. The total number of non-redundant protein identifications from each experiment was: 524 proteins by 2-D (SCX/C18) nanoflow liquid chromatography-liquid chromatography tandem mass spectrometry (nanoLC-LC MS/MS (MudPIT)); 381 proteins by nanoLC-MS/MS with gas phase fractionation by mass range selection; 390 proteins by nanoLC-MS/MS with gas phase fractionation by ion abundance selection; 898 proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins, in-gel digestion, and nanoLC-MS/MS of gel slices; and 422 proteins by isoelectric focusing of proteins, in-gel digestion and nanoLC-MS/MS of gel slices. The total number of non-redundant protein identifications in the five experiments was 1204. Combining only the two best experiments, the SDS-PAGE gel slices and the Mudpit, produces 1024 proteins identified, more than 85% of the total. Clearly, combining a Mudpit analysis with an SDS-PAGE gel slice experiment gives the greatest amount of protein identification information from a limited amount of sample.

Chemical Fractionation↗
PubMed2005

Analysis of secreted proteins from Aspergillus flavus.

MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus.

Aspergillus flavus↗
PubMed2004

Protein disulfide bond formation in the cytoplasm during oxidative stress.

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both beta4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.

Adenomatous Polyposis Coli Protein↗
PubMed2003

Proteomic characterization of the Chlamydomonas reinhardtii chloroplast ribosome. Identification of proteins unique to th e70 S ribosome.

We have conducted a proteomic analysis of the 70 S ribosome from the Chlamydomonas reinhardtii chloroplast. Twenty-seven orthologs of Escherichia coli large subunit proteins were identified in the 50 S subunit, as well as an ortholog of the spinach plastid-specific ribosomal protein-6. Several of the large subunit proteins of C. reinhardtii have short extension or insertion sequences, but overall the large subunit proteins are very similar to those of spinach chloroplast and E. coli. Two proteins of 38 and 41 kDa, designated RAP38 and RAP41, were identified from the 70 S ribosome that were not found in either of the ribosomal subunits. Phylogenetic analysis identified RAP38 and RAP41 as paralogs of spinach CSP41, a chloroplast RNA-binding protein with endoribonuclease activity. Overall, the chloroplast ribosome of C. reinhardtii is similar to those of spinach chloroplast and E. coli, but the C. reinhardtii ribosome has proteins associated with the 70 S complex that are related to non-ribosomal proteins in other species. In addition, the 30 S subunit contains unusually large orthologs of E. coli S2, S3, and S5 and a novel S1-type protein (Yamaguchi, K. et al., (2002) Plant Cell 14, 2957-2974). These additional proteins and domains likely confer functions used to regulate chloroplast translation in C. reinhardtii.

Amino Acid Sequence↗
PubMed2003

High-throughput functional affinity purification of mannose binding proteins from Oryza sativa.

We have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using alpha-D-mannose as the ligand. Bound fractions were eluted and subjected to one-dimensional electrophoresis, followed by high-performance liquid chromatography-tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue-specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high-throughput fashion.

Binding, Competitive↗
PubMed2003

Investigative proteomics: identification of an unknown plant virus from infected plants using mass spectrometry.

We describe the identification of a previously uncharacterized plant virus that is capable of infecting Nicotiana spp. and Arabidopsis thaliana. Protein extracts were first prepared from leaf tissue of uninfected tobacco plants, and the proteins were visualized with two-dimensional electrophoresis (2-DE). Matching gels were then run using protein extracts of a tobacco plant infected with tobacco mosaic virus (TMV). After visual comparison, the proteins spots that were differentially expressed in infected plant tissues were cut from the gels and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Tandem mass spectrometry data of individual peptides was searched with SEQUEST. Using this approach we demonstrated a successful proof-of-concept experiment by identifying TMV proteins present in the total protein extract. The same procedure was then applied to tobacco plants infected with a laboratory viral isolate of unknown identity. Several of the differentially expressed protein spots were identified as proteins of potato virus X (PVX), thus successfully identifying the causative agent of the uncharacterized viral infection. We believe this demonstrates that HPLC-MS/MS can be used to successfully characterize unknown viruses in infected plants.

Amino Acid Sequence↗
PubMed2003

Mass spectrometric analysis of the editosome and other multiprotein complexes in Trypanosoma brucei.

The composition of the editosome, a multi-protein complex that catalyzes uridine insertion and deletion RNA editing to produce mature mitochondrial mRNAs in trypanosomes, was analyzed by mass spectrometry. The editosomes were isolated by column chromatography, glycerol gradient sedimentation, and monoclonal antibody affinity purifications. At least 16 proteins form the catalytic core of the editosome, and additional associated proteins were identified. Analyses of mitochondrial fractions identified several non-editosome proteins and multi-protein complexes. These studies contribute to the functional annotation of T. brucei genome.

Amino Acid Sequence↗
PubMed2002

Proteomic survey of metabolic pathways in rice.

A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

Amino Acid Sequence↗
PubMed2002

Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry.

We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes were prepared from wheat (Triticum aestivum). Protein extracts were examined by one-dimensional and two-dimensional electrophoresis, followed by high performance liquid chromatography-tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.

Chromatography, High Pressure Liquid↗