Improving the identification rate of data independent label-free quantitative proteomics experiments on non-model crops: A case study on apple fruit

•A new tool to improve the identification rate of DIA data was fruitfully developed.•DIA data were linked to a DDA database on m/z-RT and on fragment mass level.•Ten times more peptides were identified in one whole apple extract sample.•In a quantitative storage experiment, the amount of identifications was doubled.•Proteins representing important pathways during fruit aging were identified.

Complex peptide extracts from non-model crops are troublesome for proper identification and quantification. To increase the identification rate of label free DIA experiments of Braeburn apple a new workflow was developed where a DDA database was constructed and linked to the DIA data. At a first level, parent masses found in DIA were searched in the DDA database based on their mass to charge ratio and retention time; at a second level, masses of fragmentation ions were compared for each of the linked spectrum. Following this workflow, a tenfold increase of peptides was identified from a single DIA run. As proof of principle, the designed workflow was applied to determine the changes during a storage experiment, achieving a two-fold identification increase in the number of significant peptides. The corresponding protein families were divided into nine clusters, representing different time profiles of changes in abundances during storage. Up-regulated protein families already show a glimpse of important pathways affecting aging during long-term storage, such as ethylene synthesis, and responses to abiotic stresses and their influence on the central metabolism.Biological significanceProteomics research on non-model crops causes additional difficulties in identifying the peptides present in, often complex, samples. This work proposes a new workflow to retrieve more identifications from a set of quantitative data, based on linking DIA and DDA data at two consecutive levels. As proof of principle, a storage experiment on Braeburn apple resulted in twice as much identified storage related peptides. Important proteins involved in central metabolism and stress are significantly up-regulated after long term storage.This article is part of a Special Issue entitled: Proteomics of non-model organisms.

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