Characterization of the orthodox Pinus occidentalis seed and pollen proteomes by using complementary gel-based and gel-free approaches

Abstract

This work presents an analysis of Pinus occidentalis pollen and seed proteomes, in which both gel-based and gel-free approaches have been used. Proteins were extracted from P. occidentalis seeds and pollen by using the TCA/acetone/phenol precipitation protocol, and protein extracts were subjected to 1- and 2-DE coupled to MALDI-TOF-TOF as well as to shotgun (nLC-LTQ-Orbitrap) analysis. All bands (1-DE) and the most abundant spots (2-DE) were excised, trypsin digested and the resulting peptides analyzed by MALDI TOF/TOF. In order to increase the proteome coverage, a gel free approach was used. Proteins were identified from mass spectra by using three different databases, including UniProtKB, NCBI and a Pinus spp. custom database [2]. The gel-based approach resulted in 42 (seeds) and 94 (pollen) protein identifications, while the shotgun approach permitted the identification of 187 (seed) and 960 (pollen) proteins. Proteins were classified based on their corresponding functional categories. In seeds, storage proteins were the most abundant ones, and some allergens and proteases were also identified. In pollen proteins related to general metabolism were the most predominant. Data are compared and discussed from a methodological and biological point of view, taking into account the particularities of the seed and pollen organs. Biological significanceIn this work we characterized P. occidentalis proteome with seeds and pollen samples implementing two complementary approaches for the analysis. We found a high content of storage protein, stress response and metabolism related proteins in the seed proteome. Similarly, in the pollen proteome we found predominant groups of proteins related to metabolism and stress response.