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

Kim Buts*, Servaas Michielssens, Maarten L.A.T.M. Hertog, Eisuke Hayakawa, Jan Cordewener, Antoine H.P. America, Bart M. Nicolai, Sebastien C. Carpentier

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

26 Citations (Scopus)


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 significance: Proteomics 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.

Original languageEnglish
Pages (from-to)31-45
Number of pages15
JournalJournal of Proteomics
Publication statusPublished - 13 Jun 2014


  • Data independent acquisition
  • Label free quantification
  • Non-model crop
  • Plant proteomics

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