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

The proteomics of orphan, unsequenced, and recalcitrant organisms is highly challenging. This is the case of the typical Mediterranean forest tree Holm oak (Quercus ilex). Proteomics has moved on quite fast from the classical 2DE-MS to shotgun or gel-free/label-free approaches, with the latter possessing a series of advantages over the gel-based ones. Before translating proteomics data into biological knowledge, a few questions as to the analysis technique itself have to be answered including its confidence in protein identification and quantification. It is important to clearly differentiate a hit from an ortholog and gene product identification, with the difference depending on the database and the confidence parameters (score, number of peptides, and coverage). With respect to quantification and for comparative purposes it is important to make sure that we are within the linear dynamic range. For that, a calibration curve based on mass spectrometry analysis of a serial dilution of the extracts should be performed. Thus, just by validating our data with the aim of improving the quality of the analysis enables us to give a correct interpretation of our results. We show a method that aims to improve the confidence in protein identification and quantification in the orphan species Q. ilex using a shotgun proteomics approach.

Differences in the triticale (X Triticosecale Wittmack) flag leaf 2-DE protein profile between varieties and nitrogen fertilization levels.

Nitrogen nutrition is one of the major factors limiting the growth and production of crop plants. Limited information on proteome changes occurring in response to nitrogen amount have been available up to now. We used 2-DE to investigate proteome differences between two triticale varieties and the changes caused by nitrogen nutrition deficit in the flag leaf tissue. Some physiological features, such as the number of tillers per plant, SPAD index, dry weight, and protein content were measured previous to the proteomic analysis. Statistical analysis identified 29 differential protein spots in the selected pairwise comparisons of experimental conditions and correlated with the expression cluster revealed by the principal component analysis. The 29 protein spots were subjected to matrix-assisted laser desorption ionization time of flight (MALDI-TOF) to deduce their possible functions. Many of these changes referred to enzymes involved in photosynthesis, metabolic pathways implicated in the balance of the energy, and redox status of the cell. This work provides a first characterization of the proteome changes that occur in response to nitrogen deficit in flag leaves of triticale plants.