Tiago S. Balbuena

Protein identification pipeline for the homology-driven proteomics.

Homology-driven proteomics is a major tool to characterize proteomes of organisms with unsequenced genomes. This paper addresses practical aspects of automated homology-driven protein identifications by LC-MS/MS on a hybrid LTQ Orbitrap mass spectrometer. All essential software elements supporting the presented pipeline are either hosted at the publicly accessible web server, or are available for free download.

Search engine processor: Filtering and organizing peptide spectrum matches

The search engine processor (SEPro) is a tool for filtering, organizing, sharing, and displaying peptide spectrum matches. It employs a novel three‐tier Bayesian approach that uses layers of spectrum, peptide, and protein logic to lead the data to converge to a single list of reliable protein identifications. SEPro is integrated into the PatternLab for proteomics environment, where an arsenal of tools for analyzing shotgun proteomic data is provided. By using the semi‐labeled decoy approach for benchmarking, we show that SEPro significantly outperforms a commercially available competitor.

Biochemical changes during seed development in Pinus taeda L.

Amino acids, polyamines, 3-indoleacetic acid (IAA), abscisic acid (ABA), buffer-soluble protein and starch contents and dry matter accumulation were analyzed in megagametophytes containing developing embryos during seed development in Pinus taeda. The highest total amino acids and polyamine contents occurred at the cotyledonary stage, followed by a significant decrease in the mature seed. Free polyamines exhibited higher levels than conjugated ones, with putrescine being the predominant type until the cotyledonary stage, and spermidine at the mature seed stage. IAA content increased continually from the globular stage reaching the maximum at the cotyledonary stage, followed by a decrease in the mature seed. The highest ABA level occurred at the globular stage, followed by a continuous reduction until stabilization at the pre-cotyledonary stage. Buffer-soluble protein and starch contents, and dry matter increased progressively during development, reaching their maximum values at the mature stage.

IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis

The aim of this work was to study morphological and biochemical aspects during zygotic embryogenesis in O. catharinensis, by measuring changes in the endogenous concentrations of proteins, amino acids, polyamines (PAs), indole-3-acetic acid (IAA) and abscisic acid (ABA). Buffer-soluble and insoluble protein contents were determined by spectrometry, and amino acids, PAs, IAA and ABA concentrations were determined by high performance liquid chromatography. Total amino acid accumulation, predominantly asparagine, occurred when the embryo showed completely developed cotyledons, with posterior reduction in the mature embryo. This decrease in total amino acid concentration in the mature embryo may result from their use in storage␣as well as for LEA protein synthesis. Free putrescine (Put) concentration decreased, while free spermine (Spm) increased during embryo development. This suggest a role for Put in the initial phases of embryogenesis when high rates of cell division occur, while elevated concentration of Spm are essential from the middle to the end of embryo development, when growth is mainly due to cell elongation. An IAA peak in zygotic embryos occurred during initial development, suggesting a link between growth and cellular division as well as with the establishment of bilateral symmetry. ABA concentration declined during initial stages of development then increased at the mature embryo stage, suggesting a possible relationship with dormancy and recalcitrance characteristics. Our results show that changes in the phytohormones (IAA, ABA and PAs) concentrations in combination with amino acids are likely important factors determining the developmental stages of O.␣catharinensis zygotic embryos.

Separating the Wheat from the Chaff: Unbiased Filtering of Background Tandem Mass Spectra Improves Protein Identification

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.

Proteome analysis of cold acclimation in sunflower.

Cold acclimation is the phenomenon in which plants are exposed to low, but nonfreezing, temperatures before exposure to drastic temperatures. To investigate how sunflower plants adjust their metabolism during cold treatment, a comparative proteomic approach, based on spectral counting data, was adopted to identify differentially expressed proteins in leaves of freezing susceptible (Hopi) and tolerant (PI 543006 and BSD-2-691) lines after cold acclimation. In total 718, 675, and 769 proteins were confidently identified by tandem mass spectrometry in Hopi, PI 543006, and BSD-2-691 sunflower lines. Tolerant lines PI 543006 and BSD-2-691 showed the highest number of differentially expressed proteins, as 43, 72, and 168 proteins changed their expression in Hopi, PI 543006, and BSD-2-691 sunflower lines, respectively, at 95% confidence. Cold-responsive proteins were mostly involved in metabolism, protein synthesis, energy, and defense processes in all sunflower lines studied. Hierarchical clustering of all differentially expressed proteins resulted in the characterization of 14 different patterns of expression across Hopi, PI 543006, and BSD-2-691 and indicated that tolerant lines showed different proteome responses to cold acclimation.

Next-generation sequencing-based transcriptomic and proteomic analysis of the common reed, Phragmites australis (Poaceae), reveals genes involved in invasiveness and rhizome specificity

PREMISE OF THE STUDY The common reed (Phragmites australis), one of the most widely distributed of all angiosperms, uses its rhizomes (underground stems) to invade new territory, making it one of the most successful weedy species worldwide. Characterization of the rhizome transcriptome and proteome is needed to identify candidate genes and proteins involved in rhizome growth, development, metabolism, and invasiveness. METHODS We employed next-generation sequencing technologies including 454 and Illumina platforms to characterize the reed rhizome transcriptome and used quantitative proteomics techniques to identify the rhizome proteome. KEY RESULTS Combining 336514 Roche 454 Titanium reads and 103350802 Illumina paired-end reads in a de novo hybrid assembly yielded 124450 unique transcripts with an average length of 549 bp, of which 54317 were annotated. Rhizome-specific and differentially expressed transcripts were identified between rhizome apical tips (apical meristematic region) and rhizome elongation zones. A total of 1280 nonredundant proteins were identified and quantified using GeLC-MS/MS based label-free proteomics, where 174 and 77 proteins were preferentially expressed in the rhizome elongation zone and apical tip tissues, respectively. Genes involved in allelopathy and in controlling development and potentially invasiveness were identified. CONCLUSIONS In addition to being a valuable sequence and protein data resource for studying plant rhizome species, our results provide useful insights into identifying specific genes and proteins with potential roles in rhizome differentiation, development, and function.

Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia

Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes, oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed.

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

BACKGROUND AND AIMS Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination. METHODS Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC. KEY RESULTS During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied. CONCLUSIONS The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.

The response of Asterochloris erici (Ahmadjian) Skaloud et Peksa to desiccation: a proteomic approach

The study of desiccation tolerance of lichens, and of their chlorobionts in particular, has frequently focused on the antioxidant system that protects the cell against photo-oxidative stress during dehydration/rehydration cycles. In this study, we used proteomic and transcript analyses to assess the changes associated with desiccation in the isolated phycobiont Asterochloris erici. Algae were dried either slowly (5-6 h) or rapidly (<60 min), and rehydrated after 24 h in the desiccated state. To identify proteins that accumulated during the drying or rehydration processes, we employed two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with individual protein identification using trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analyses revealed that desiccation caused an increase in relative abundance of only 11-13 proteins, regardless of drying rate, involved in glycolysis, cellular protection, cytoskeleton, cell cycle, and targeting and degradation. Transcripts of five Hsp90 and two β-tubulin genes accumulated primarily at the end of the dehydration process. In addition, transmission electron microscopy (TEM) images indicate that ultrastructural cell injuries, perhaps resulting from physical or mechanical stress rather than metabolic damage, were more intense after rapid dehydration. This occurred with no major change in the proteome. These results suggest that desiccation tolerance of A. erici is achieved by constitutive mechanisms.