Nebraska U FARRP - Cashew Proteome and Method Development

Proteomic Analysis of Oil-Roasted Cashew Using a Customized Allergen-Focused Protein Database
Data License: CC BY 4.0 | ProteomeXchange: PXD030375 | doi:
  • Organism: Anacardium occidentale
  • Instrument: Q Exactive Plus
  • SpikeIn: No
  • Keywords: Cashew, Proteomics, Proteogenomics, Oil roasting, Allergen
  • Lab head: Melanie Downs Submitter: Shimin Chen
Cashew is one of the most prevalent causes of tree nut allergies. However, the cashew proteome is far from complete, which limits the quality of peptide identification in mass spectrometric analyses. In this study, bioinformatics tools were utilized to construct a customized cashew protein database and improve sequence quality for proteins of interest, based on a publicly available cashew genome database. As a result, two additional isoforms for cashew 2S albumins and five other isoforms for cashew 11S proteins were identified, along with several other potential allergens. Using the optimized protein database, the protein profiles of cashew nuts subjected to different oil-roasting conditions (138 °C and 166 °C for 2-10 minutes) were analyzed using discovery LC-MS/MS analysis. The results showed that cashew 2S protein is most heat-stable, followed by 11S and 7S proteins, though protein isoforms might be affected differently. Preliminary target peptide selection indicated that out of the 29 potential targets, 18 peptides were derived from the newly developed database. In the evaluation of thermal processing effects on cashew proteins, several Maillard reaction adducts were also identified. The cashew protein database developed in this study allows for comprehensive analyses of cashew proteome and development of high-quality allergen detection method.
Sample Description
Raw cashew nut halves (100 g) were oil-roasted in 1 L soybean oil using a kitchen-scale fryer at two different temperatures (139 °C and 166 °C) for different time durations (2, 4, 8, and 10 minutes). The temperature fluctuation, measured by a digital thermometer, was +/- 5 °C over the course of roasting. Approximately 25 g of cashew halves were sampled at each time point. All cashew halves were placed on a paper towel to remove excess oil. Samples were then ground and stored at -20 °C. Cashew nuts were ground and extracted at a 1:20 sample-to-buffer ratio (w/v, 0.07 g sample: 1.4 mL buffer) using 50 mM Tris-HCl, pH 8.6, with 6 M urea and 20 mM DTT. The samples were incubated in a 60 °C shaking water bath for 30 min, followed by centrifugation at 13000 g for 10 min. Ten-fold diluted extracts (containing approximately 10 µg protein) were taken for in-solution tryptic digestion. The diluted extract (21 µL) was mixed with 50 mM AMBC (30 µL) and 100 mM DTT (3 µL) and incubated at 95 ºC for 5 minutes. Upon cooling, 100 mM IAA (6 µL) was added to samples, which were then placed in the dark at room temperature for 20 minutes. After alkylation, samples were digested with trypsin at 1:100 protein-to-trypsin ratio (w/w) at 37 ºC for 3 hours, then added with the same amount of trypsin for overnight digestion (at least 12 hours). The digested samples were desalted using Pierce C18 spin columns following the manufacturer’s instructions. Samples were then dried in a vacuum evaporator and stored at -20 °C. Prior to injection, samples were resuspended in 40 µL 0.1% formic acid and 3% acetonitrile in HPLC-grade water.
Created on 12/13/21, 4:20 PM
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20191218_Oil-roasted-cashew_FinalDB-major prot 16:19:4910305015036