<?xml version="1.0" encoding="UTF-8"?><xml><records><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors></contributors><titles><title><style face="normal" font="default" size="100%">Improving the quality of protein identification in non-model species. Characterization of Quercus ilex seed and Pinus radiata needle proteomes by using SEQUEST and custom databases</style></title><secondary-title><style face="normal" font="default" size="100%">Journal of Proteomics</style></secondary-title></titles><dates><year><style  face="normal" font="default" size="100%">2014</style></year></dates><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">Nowadays the most used pipeline for protein identification consists in the comparison of the MS/MS spectra to reference databases. Search algorithms compare obtained spectra to an in silico digestion of a sequence database to find exact matches. In this context, the database has a paramount importance and will determine in a great deal the number of identifications and its quality, being this especially relevant for non-model plant species. Using a single Viridiplantae database (NCBI, UniProt, TAIR) is not the best choice for non-model species since they are underrepresented in databases resulting in poor identification rates. We demonstrate how it is possible to improve the rate and quality of identifications in two orphan species, Quercus ilex and Pinus radiata, by using SEQUEST and a combination of public (Viridiplantae NCBI, UniProt) and a custom-built specific database which contained 593,294 and 455,096 peptide sequences (Quercus and Pinus, respectively). These databases were built after gathering and processing (trimming, contiging, 6-frame translation) publicly available RNA sequences, mostly ESTs and NGS reads. A total of 149 and 1533 proteins were identified from Quercus seeds and Pinus needles, representing a 3.1- or 1.5-fold increase in the number of protein identifications and scores compared to the use of a single database. Since this approach greatly improves the identification rate, and is not significantly more complicated or time consuming than other approaches, we recommend its routine use when working with non-model species. BIOLOGICAL SIGNIFICANCE In this work we demonstrate how the construction of a custom database (DB) gathering all available RNA sequences and its use in combination with Viridiplantae public DBs (NCBI, UniProt) significantly improve protein identification when working with non-model species. Protein identification rate and quality is higher to those obtained in routine procedures based on using only one database (commonly Viridiplantae from NCBI), as we demonstrated analyzing Quercus seeds and Pine needles. The proposed approach based on the building of a custom database is not difficult or time consuming, so we recommend its routine use when working with non-model species. This article is part of a Special Issue entitled: Proteomics of non-model organisms.</style></abstract></record><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors><authors><author><style face="normal" font="default" size="100%">Sghaier-Hammami, Besma</style></author><author><style face="normal" font="default" size="100%">Valero-Galvàn, José</style></author><author><style face="normal" font="default" size="100%">Romero-rodríguez, Mª Cristina</style></author><author><style face="normal" font="default" size="100%">Navarro-Cerrillo, Rafael Mª Ma</style></author><author><style face="normal" font="default" size="100%">Abdelly, Chedly</style></author><author><style face="normal" font="default" size="100%">Jorrín-novo, Jesús</style></author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Physiological and proteomics analyses of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) responses to Phytophthora cinnamomi</style></title><secondary-title><style face="normal" font="default" size="100%">Plant Physiology and Biochemistry</style></secondary-title></titles><keywords><keyword><style  face="normal" font="default" size="100%">Gene Expression Regulation</style></keyword><keyword><style  face="normal" font="default" size="100%">Holm oak decline</style></keyword><keyword><style  face="normal" font="default" size="100%">Holm oak proteomics</style></keyword><keyword><style  face="normal" font="default" size="100%">Phytophthora</style></keyword><keyword><style  face="normal" font="default" size="100%">phytophthora cinnamomi</style></keyword><keyword><style  face="normal" font="default" size="100%">Phytophthora: physiology</style></keyword><keyword><style  face="normal" font="default" size="100%">Plant</style></keyword><keyword><style  face="normal" font="default" size="100%">Plant Proteins</style></keyword><keyword><style  face="normal" font="default" size="100%">Plant Proteins: genetics</style></keyword><keyword><style  face="normal" font="default" size="100%">Plant Proteins: metabolism</style></keyword><keyword><style  face="normal" font="default" size="100%">Plant: genetics</style></keyword><keyword><style  face="normal" font="default" size="100%">Plant: physiology</style></keyword><keyword><style  face="normal" font="default" size="100%">Proteomics</style></keyword><keyword><style  face="normal" font="default" size="100%">Proteomics: methods</style></keyword><keyword><style  face="normal" font="default" size="100%">Quercus</style></keyword><keyword><style  face="normal" font="default" size="100%">Quercus ilex</style></keyword><keyword><style  face="normal" font="default" size="100%">Quercus: microbiology</style></keyword></keywords><dates><year><style  face="normal" font="default" size="100%">2013</style></year><pub-dates><date><style  face="normal" font="default" size="100%">2013///</style></date></pub-dates></dates><urls><web-urls><url><style face="normal" font="default" size="100%">http://www.sciencedirect.com/science/article/pii/S0981942813002611http://dx.doi.org/10.1016/j.plaphy.2013.06.030http://www.ncbi.nlm.nih.gov/pubmed/23962806</style></url></web-urls></urls><volume><style face="normal" font="default" size="100%">71</style></volume><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">Phytophthora cinnamomi is one of the agents that trigger the decline syndrome in Quercus spp., this being a serious threat to Mediterranean Holm oak forest sustainability and reforestation programs. Quercus ilex responses to Phytophthora cinnamomi have been studied in one-year olds seedlings from two Andalucía provenances, assessing the physiological water status and photosynthesis-related parameters. Upon inoculation with mycelium a reduction in water content, chlorophyll fluorescence, stomatal conductance and gas exchange was observed along a 90 days post inoculation period in both provenances. The reduction was higher in the most susceptible (SSA) provenance, than in the most tolerant (PCO), being these typical plant responses to drought stress. Leaf protein profiles were analyzed in non-inoculated and inoculated seedlings from the two provenances by using a 2-DE coupled to MS proteomics strategy. Ninety seven proteins changing in abundance in response to the inoculation were successfully identified after MALDI–TOF–TOF analyses. The largest group of variable identified proteins were chloroplasts ones, and they were involved in the photosynthesis, Calvin cycle and carbohydrate metabolism. It was noted that a general tendency was a decrease in the protein abundance as a consequence of the inoculation, being it less accused in the least susceptible, the Northern provenance (PCO), than in the most susceptible, the Southern provenance (SSA). This trend is clearly manifested in photosynthesis, amino acid metabolism and stress/defence proteins. On the contrary, some proteins related to starch biosynthesis, glycolysis and stress related peroxiredoxin showed an increase upon inoculation. These changes in protein abundance were correlated to the estimated physiological parameters and have been frequently observed in plants subjected to drought stress.</style></abstract><notes><style face="normal" font="default" size="100%">From Duplicate 1 (Physiological and proteomics analyses of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) responses to Phytophthora cinnamomi - Sghaier-Hammami, Besma; Valero-Galvàn, José; Romero-rodríguez, Mª Cristina; Navarro-Cerrillo, Rafael Mª; Abdelly, Chedly; Jorrín-novo, Jesús)From Duplicate 1 (Physiological and proteomics analyses of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) responses to Phytophthora cinnamomi - Sghaier-Hammami, Besma; Valero-Galvàn, José; Romero-rodríguez, Mª Cristina; Navarro-Cerrillo, Rafael Mª; Abdelly, Chedly; Jorrín-novo, Jesús)The following values have no corresponding Zotero field:&lt;br/&gt;publisher: Elsevier Masson SAS&lt;br/&gt;accession-num: 23962806</style></notes></record><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors></contributors><titles><title><style face="normal" font="default" size="100%">Physiological and proteomics analyses of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) responses to Phytophthora cinnamomi</style></title><secondary-title><style face="normal" font="default" size="100%">Plant Physiology and Biochemistry</style></secondary-title></titles><dates><year><style  face="normal" font="default" size="100%">2013</style></year></dates><publisher><style face="normal" font="default" size="100%">Elsevier Masson SAS</style></publisher><volume><style face="normal" font="default" size="100%">71</style></volume><pages><style face="normal" font="default" size="100%">--</style></pages><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">Phytophthora cinnamomi is one of the agents that trigger the decline syndrome in Quercus spp., this being a serious threat to Mediterranean Holm oak forest sustainability and reforestation programs. Quercus ilex responses to Phytophthora cinnamomi have been studied in one-year olds seedlings from two Andalucía provenances, assessing the physiological water status and photosynthesis-related parameters. Upon inoculation with mycelium a reduction in water content, chlorophyll fluorescence, stomatal conductance and gas exchange was observed along a 90 days post inoculation period in both provenances. The reduction was higher in the most susceptible (SSA) provenance, than in the most tolerant (PCO), being these typical plant responses to drought stress. Leaf protein profiles were analyzed in non-inoculated and inoculated seedlings from the two provenances by using a 2-DE coupled to MS proteomics strategy. Ninety seven proteins changing in abundance in response to the inoculation were successfully identified after MALDI–TOF–TOF analyses. The largest group of variable identified proteins were chloroplasts ones, and they were involved in the photosynthesis, Calvin cycle and carbohydrate metabolism. It was noted that a general tendency was a decrease in the protein abundance as a consequence of the inoculation, being it less accused in the least susceptible, the Northern provenance (PCO), than in the most susceptible, the Southern provenance (SSA). This trend is clearly manifested in photosynthesis, amino acid metabolism and stress/defence proteins. On the contrary, some proteins related to starch biosynthesis, glycolysis and stress related peroxiredoxin showed an increase upon inoculation. These changes in protein abundance were correlated to the estimated physiological parameters and have been frequently observed in plants subjected to drought stress.</style></abstract><accession-num><style face="normal" font="default" size="100%">23962806</style></accession-num><notes><style face="normal" font="default" size="100%">From Duplicate 1 (Physiological and proteomics analyses of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) responses to Phytophthora cinnamomi - Sghaier-Hammami, Besma; Valero-Galvàn, José; Romero-rodríguez, Mª Cristina; Navarro-Cerrillo, Rafael Mª; Abdelly, Chedly; Jorrín-novo, Jesús)</style></notes><research-notes><style face="normal" font="default" size="100%">From Duplicate 1 (Physiological and proteomics analyses of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) responses to Phytophthora cinnamomi - Sghaier-Hammami, Besma; Valero-Galvàn, José; Romero-rodríguez, Mª Cristina; Navarro-Cerrillo, Rafael Mª; Abdelly, Chedly; Jorrín-novo, Jesús)</style></research-notes></record><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors></contributors><titles><title><style face="normal" font="default" size="100%">Simple, rapid and reliable methods to obtain high quality RNA and genomic DNA from Quercus ilex L. leaves suitable for molecular biology studies</style></title><secondary-title><style face="normal" font="default" size="100%">Acta Physiologiae Plantarum</style></secondary-title></titles><dates><year><style  face="normal" font="default" size="100%">2011</style></year></dates><volume><style face="normal" font="default" size="100%">34</style></volume><pages><style face="normal" font="default" size="100%">793-805</style></pages><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">Isolation of high-quality RNA and genomic DNA (gDNA) from many samples is a necessary step before accomplishing molecular biology studies. The particular composition of Quercus ilex leaves, specially hard and rich in cell wall material, polyphenolics and secondary metabolites, usually results in preparations contaminated with non-nucleic acid compounds. Although many methods have been developed, each case of study demands a protocol adapted to the speciﬁc plant sample and the pursued research objectives. We have evaluated several protocols to establish the methodology that best suited to our current genetic and molecular studies on Q. ilex. Our priority was to select the simplest methods reducing the plant starting material and the time employed, without compromising yield, quality and integrity of the isolated nucleic acids. Our results point to two protocols based on silicamembrane puriﬁcation, as the most convenient for Q. ilex leaf tissue, and both procedures are greatly improved by adding insoluble polyvinyl polypyrrolidone during the isolation process. The protocols optimized here can be completed at the microfuge scale and allow a researcher to process 48 samples in 1 h, producing high quality preparations suitable for the routinely molecular biology applications with higher efﬁciency than other more labour and time-consuming protocols</style></abstract></record></records></xml>