Occurrence of Hydroxyproline in Proteomes of Higher Plants

Authors

Olivia Huffman, University of Nebraska-LincolnFollow

First Advisor

Joseph Baumert

Second Advisor

Philip Johnson

Date of this Version

11-2022

Citation

Huffman, O. K. Occurrence of Hydroxyproline in Proteomes of Higher Plants. 2022.

Comments

A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Food Science and Technology, Under the Supervision of Professors Joseph L. Baumert and Philip Johnson. Lincoln, Nebraska: November 2022

Copyright © 2022 Olivia K. Huffman

Abstract

Food allergies affect millions of individuals across the United States and worldwide. Peanut allergies are among the most severe food allergies because of their potentially life-threatening symptoms and lifelong persistence. Potent peanut allergen, Ara h 2, is known to contain an amino acid motif containing the posttranslational modification, hydroxyproline (HyP). HyP is associated with immunogenic response when present both in Ara h 2 and in timothy grass pollen allergen, Phl p 1. To further explore the presence of HyP in higher plants and specifically to investigate its potential presence in commonly allergenic plants, a study of 26 plant seeds was conducted using hydrolyzed amino acid analysis (HAA) and data-dependent acquisition (DDA) through liquid chromatography and tandem mass spectrometry (LCMS/MS). Curated protein databases allowed for database searches using PEAKS software. Samples for which no database could be procured were analyzed using de novo sequencing. Results showed detection of HyP in 25 out of 26 plant seed samples. HyP sites were classified into one of four tiers based on the quality of the database used to identify a given site. To further refine the identified HyP sites and to increase confidence in their position and identity, a manual analysis approach was performed in addition to software analyses. This approach was successful in reducing the number of sites for each sample as well as increasing the confidence of those sites. Peanut presented as a clear outlier in the number of HyP sites in software and de novo analyses both before and after refinement by manual analysis. The results indicate that species across Viridiplantae possess the machinery to perform prolyl hydroxylation. Furthermore, these data indicate that peanut is unique in its quantity of HyP sites even when normalized to the total number of proline residues.

Advisors: Joseph L. Baumert and Philip Johnson