Comparing and integrating TMT-SPS-MS3 and label-free quantitative approaches for proteomics scrutiny in recalcitrant Mango (Mangifera indica L.) peel tissue during postharvest period.

Despite substantial advances in the use of proteomic technologies, their widespread application in fruit tissues of non-model and recalcitrant species remains limited. This hampers the understanding of critical molecular events during the postharvest period of fleshy tropical fruits. Therefore, we evaluated label-free quantitation (LFQ) and TMT-SPS-MS3 (TMT) approaches to analyse changes in the protein profile of mango peels during postharvest period. We compared two extraction methods (phenol and chloroform/methanol) and two peptide fractionation schemes (SCX and HPRP). We accurately identified 3065 proteins, of which, 1492 were differentially accumulated over at 6 days after harvesting (DAH). Both LFQ and TMT approaches share 210 differential proteins including cell wall proteins associated with fruit softening, as well as aroma and flavour-related proteins, which were increased during postharvest period. The phenolic protein extraction and the high-pH reverse-phase peptide fractionation was the most effective pipeline for relative quantification. Nevertheless, the information provided by the other tested strategies was significantly complementary. Besides, LFQ spectra allowed us to track down intact N-glycopeptides corroborating N-glycosylations on the surface of a desiccation-related protein. This work represents the largest proteomic comparison of mango peels during postharvest period made so far, shedding light on the molecular foundation of edible fruit during ripening.

Proteometabolomic Analysis Reveals Molecular Features Associated with Grain Size and Antioxidant Properties amongst Chickpea (Cicer arietinum L.) Seeds Genotypes.

Legumes are an essential source of nutrients that complement energy and protein requirements in the human diet. They also contribute to the intake of bioactive compounds such as polyphenols, whose content can vary depending on cultivars and genotypes. We conducted a comparative proteomics and metabolomics study to determine if there were significant variations in relevant nutraceutical compounds in the five genotypes of Kabuli-type chickpea grains. We performed an isobaric tandem mass tag (TMT) couple to synchronous precursor selection (SPS)-MS3 method along with a targeted and untargeted metabolomics approach based on accurate mass spectrometry. We observed an association between the overproduction of proteins involved in starch, lipid, and amino acid metabolism with gibberellin accumulation in large grains. In contrast, we visualized the over-accumulation of proteins associated with water deprivation in small grains. It was possible to visualize in small grains the over-accumulation of some phenolics such as vanillin, salicylic acid, protocatechuic acid, 4-coumaric acid, 4-hydroxybenzoic acid, vanillic acid, ferulic acid, and kaempferol 3-O-glucoside as well as the amino acid l-phenylalanine. The activated phenolic pathway was associated with the higher antioxidant capacity of small grains. Small grains consumption could be advantageous due to their nutraceutical properties.

Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus: An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress.

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.