Optimizing Shotgun Proteomics Analysis for a Confident Protein Identification and Quantitation in Orphan Plant Species: The Case of Holm Oak (Quercus ilex)

Gómez-Gálvez, Isabel; Sánchez-Lucas, Rosa; San-Eufrasio, Bonoso; Francisco, Luis; Maldonado‐Alconada, Ana M.; Fuentes-Almagro, Carlos; Castillejo, María Ángeles

doi:10.1007/978-1-0716-0528-8_12

Cited by 8 publications

(6 citation statements)

References 10 publications

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“…Grain protein was extracted according to the trichloroacetic acid (TCA)/acetone–phenol protocol 18 and its concentration determined with the Bradford method (BioRad, Hercules, CA, USA), using bovine serum albumin (BSA) as standard 19 . For protein sample cleaning, 60 µg of BSA protein equivalent from each biological replicate of each treatment was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the only resulting band digested with trypsin (12.5 ng µL −1 ) (Sequencing grade, Promega, Madison, WI, USA) 19 . A shotgun (Liquid Chromatography with tandem mass spectrometry, LC–MS/MS) analysis was then performed according to Castillejo et al 20 .…”

Section: Methodsmentioning

confidence: 99%

Combining P and Zn fertilization to enhance yield and grain quality in maize grown on Mediterranean soils

Sánchez‐Rodríguez

Rey

Nechate-Drif

et al. 2021

Sci Rep

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The main aim of this study was to elucidate the effect of individual and joint fertilization with P and Zn on maize plants grown on typical Mediterranean soils with a limited Zn availability. For this purpose, we examined the effects of P and Zn fertilization individually and in combination on growth, yield and grain protein content in maize grown in pots filled with three different Mediterranean soils (LCV, FER and INM). Phosphorus and Zn translocation to grain was impaired, and aboveground dry matter and yield at harvest reduced by 8–85% (LCV and FER), in plants treated with Zn or P alone relative to unfertilized (control) plants. In contrast, joint fertilization with P and Zn enhanced translocation of these nutrients to grain and significantly increased aboveground dry matter (30% in LCV, 50% in FER and 250% in INM) and grain Zn availability in comparison with control plants. Also, joint application of both nutrients significantly increased grain P (LCV) and Zn (LCV and FER) use efficiency relative P and Zn, respectively, alone. Yield was increased between 31% in LCV and 121% in FER relative to control plants, albeit not significantly. Fertilization with P or Zn significantly influenced the abundance of specific proteins affecting grain quality (viz., storage, lys-rich and cell wall proteins), which were more abundant in mature grains from plants fertilized with Zn alone and, to a lesser extent, P + Zn. Sustainable strategies in agriculture should consider P–Zn interactions in maize grown on soils with a limited availability of Zn, where Zn fertilization is crucial to ensure grain quality.

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Section: Methodsmentioning

confidence: 99%

Combining P and Zn fertilization to enhance yield and grain quality in maize grown on Mediterranean soils

Sánchez‐Rodríguez

Rey

Nechate-Drif

et al. 2021

Sci Rep

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“…A number of proteomic studies have used 1D and 2D gel-based analysis to investigate drought tolerance in this species [11,12,23]. In addition, recent studies have addressed various aspects of its biology by using shotgun (LC-MS/MS) proteomic analysis [19,21,24]. In addition, species-specific improved databases, such as the recently compiled holm oak transcriptome database [16,17], and other sequenced Quercus species databases, such as those for Q. robur [25] and Q. suber [26], have substantially expanded available knowledge of holm oak biology.…”

Section: Introductionmentioning

confidence: 99%

Proteomics Data Analysis for the Identification of Proteins and Derived Proteotypic Peptides of Potential Use as Putative Drought Tolerance Markers for Quercus ilex

San-Eufrasio

Bigatton

Guerrero‐Sanchez

et al. 2021

IJMS

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Drought is one of the main causes of mortality in holm oak (Quercus ilex) seedlings used in reforestation programs. Although this species shows high adaptability to the extreme climate conditions prevailing in Southern Spain, its intrinsic genetic variability may play a role in the differential response of some populations and individuals. The aim of this work was to identify proteins and derived proteotypic peptides potentially useful as putative markers for drought tolerance in holm oak by using a targeted post-acquisition proteomics approach. For this purpose, we used a set of proteins identified by shotgun (LC-MSMS) analysis in a drought experiment on Q. ilex seedlings from four different provenances (viz. the Andalusian provinces Granada, Huelva, Cadiz and Seville). A double strategy involving the quantification of proteins and target peptides by shotgun analysis and post-acquisition data analysis based on proteotypic peptides was used. To this end, an initial list of proteotypic peptides from proteins highly represented under drought conditions was compiled that was used in combination with the raw files from the shotgun experiment to quantify the relative abundance of the fragment’s ion peaks with the software Skyline. The most abundant peptides under drought conditions in at least two populations were selected as putative markers of drought tolerance. A total of 30 proteins and 46 derived peptides belonging to the redox, stress-related, synthesis,-folding and degradation, and primary and secondary metabolism functional groups were thus identified. Two proteins (viz., subtilisin and chaperone GrpE protein) were found at increased levels in three populations, which make them especially interesting for validation drought tolerance markers in subsequent experiments.

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“…Samples containing 70 μg of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide for protein cleaning according to Romero-Rodríguez et al (2019) . The only resulting band was excised and digested with trypsin and the peptides thus obtained were dissolved in 50 μl of a 4% acetonitrile/0.25% formic acid mixture for loading into an Ultimate 3,000 nano-LC-MS-UHPLC- Orbitrap Fusion instrument from Thermo Fisher Scientific (Waltham, MA, United States; Gómez-Gálvez et al, 2020 ). MS/MS data were processed with the software Proteome Discoverer v. 2.3, also from Thermo Fischer Scientific, and identified by using the SEQUEST algorithm against the species-specific database of Q. ilex developed from the transcriptome ( Guerrero-Sánchez et al, 2017 , 2019 , 2021 ), using the setting reported by San-Eufrasio et al (2021) .…”

Section: Methodsmentioning

confidence: 99%

Identification of Proteases and Protease Inhibitors in Seeds of the Recalcitrant Forest Tree Species Quercus ilex

et al. 2022

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Proteases and protease inhibitors have been identified in the recalcitrant species Quercus ilex using in silico and wet methods, with focus on those present in seeds during germination. In silico analyses showed that the Q. ilex transcriptome database contained 2,240 and 97 transcripts annotated as proteases and protease inhibitors, respectively. They belonged to the different families according to MEROPS,1 being the serine and metallo ones the most represented. The data were compared with those previously reported for other Quercus species, including Q. suber, Q. lobata, and Q. robur. Changes in proteases and protease inhibitors alongside seed germination in cotyledon and embryo axis tissues were assessed using proteomics and in vitro and in gel activity assays. Shotgun (LC–MSMS) analysis of embryo axes and cotyledons in nonviable (NV), mature (T1) and germinated (T3) seeds allowed the identification of 177 proteases and 12 protease inhibitors, mostly represented by serine and metallo types. Total protease activity, as determined by in vitro assays using azocasein as substrate, was higher in cotyledons than in embryo axes. There were not differences in activity among cotyledon samples, while embryo axis peaked at germinated T4 stage. Gel assays revealed the presence of protease activities in at least 10 resolved bands, in the Mr range of 60–260 kDa, being some of them common to cotyledons and embryo axes in either nonviable, mature, and germinated seeds. Bands showing quantitative or qualitative changes upon germination were observed in embryo axes but not in cotyledons at Mr values of 60–140 kDa. Proteomics shotgun analysis of the 10 bands with protease activity supported the results obtained in the overall proteome analysis, with 227 proteases and 3 protease inhibitors identified mostly represented by the serine, cysteine, and metallo families. The combined use of shotgun proteomics and protease activity measurements allowed the identification of tissue-specific (e.g., cysteine protease inhibitors in embryo axes of mature acorns) and stage-specific proteins (e.g., those associated with mobilization of storage proteins accumulated in T3 stage). Those proteins showing differences between nonviable and viable seeds could be related to viability, and those variables between mature and germinated could be associated with the germination process. These differences are observed mostly in embryo axes but not in cotyledons. Among them, those implicated in mobilization of reserve proteins, such as the cathepsin H cysteine protease and Clp proteases, and also the large number of subunits of the CNS and 26S proteasome complex differentially identified in embryos of the several stages suggests that protein degradation via CNS/26S plays a major role early in germination. Conversely, aspartic proteases such as nepenthesins were exclusively identified in NV seeds, so their presence could be used as indicator of nonviability.

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Optimizing Shotgun Proteomics Analysis for a Confident Protein Identification and Quantitation in Orphan Plant Species: The Case of Holm Oak (Quercus ilex)

Cited by 8 publications

References 10 publications

Combining P and Zn fertilization to enhance yield and grain quality in maize grown on Mediterranean soils

Combining P and Zn fertilization to enhance yield and grain quality in maize grown on Mediterranean soils

Proteomics Data Analysis for the Identification of Proteins and Derived Proteotypic Peptides of Potential Use as Putative Drought Tolerance Markers for Quercus ilex

Identification of Proteases and Protease Inhibitors in Seeds of the Recalcitrant Forest Tree Species Quercus ilex

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