Identification of bitter pit protein markers in Malus domestica using differential in-gel electrophoresis (DIGE) and LC–MS/MS

• Bitter pit (BP) induces a deep change in the physiological condition of tissues.
• Pathogenesis-related (PR) proteins from several types are overexpressed in BP.
• Mald1 and Mald2 may increase allergenicity on apple without visual BP symptoms.
• Healthy proteins contribute to ethylene, glyco-protein and lipid production.

Bitter pit is a physiological disorder that occurs in apple, pear and quince and has long been associated with calcium uptake or lack thereof. In the present study, pooled biological Malus domestica proteins were collected from healthy and naturally occurring bitter pit fruit. Protein samples (bitter pit and healthy) were analyzed with differential in-gel electrophoresis (DIGE) and SameSpots software was used to compare gel spots by intensity. Identified spots (p < 0.05) were spot picked and trypsin digested. Peptides were separated by liquid chromatography (LC) and submitted to LTQ-Orbitrap mass spectrometer to infer protein identification. A total of 200 ± 5 protein spots were detected, 41 spots classified as having p < 0.05 and were successfully identified by their peptide sequence listed in an online M. domestica database. Thirteen spots were identified as having p < 0.05 and a minimum 2-fold change. Several pathogenesis-related (PR) proteins belonging to three PR families, PR-5 (10.3-fold), PR-8/chitinase (35.5-fold) and PR-10 (6.8-fold) were upregulated in bitter pit sampled tissues. Three proteins involved in several metabolic processes including ethylene biosynthesis (3.3-fold), glycosyltransferase reactions in metabolism (2.3-fold) and metal biding (11.1-fold) were found to be overly expressed in healthy sampled tissues. This research provides a significant advance in the knowledge of protein expression alterations occurring in bitter pit in comparison with sound tissues.