Development and Validation of MRM Methods to Quantify Protein Isoforms of Polyphenol Oxidase in Loquat Fruits

Abstract

Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using external calibrants.

Discordance between protein and transcript levels detected by selected reaction monitoring

“…Nevertheless, protein identification in model organisms still has advantages because of the existence of more accurate amino acid sequences. On the other hand, SRM analysis can be applied to non-model plants, 6,11,24 if the targeted peptides are selected. This indicates that complete genome sequences of the organisms of interest are not required.…”

“…Therefore, SRM analysis enables individual quantification of not only homologous proteins but also proteins in very low abundances. 6 …”

“…4 The iTRAQ approach, specifically, has been adapted to compare and quantify proteins in various plant species. [5][6][7][8][9][10] Digested peptides in each protein sample are labeled with different iTRAQ reagents and samples are then pooled. Therefore, samples can be analyzed by LC/MS at the same time.…”

Multi-omics Frontiers in Algal Research: Techniques and Progress to Explore Biofuels in the Postgenomics World

“…PRM is thus emerging as an efficient tool over other targeted approaches. However, till date, MS-based target validation in microalgae is yet to be tried, while very limited studies have been performed in plants (Majovsky et al, 2014;Martinez-Marquez et al, 2013).…”

Quantitative changes in proteins responsible for flavonoid and anthocyanin biosynthesis in strawberry fruit at different ripening stages: A targeted quantitative proteomic investigation employing multiple reaction monitoring

“…This platform opens exciting possibilities in plant biotechnological and biological applications, and makes the quantitative analysis of a whole proteome by MRM a concrete possibility. Until now, there have only been a few reports available on the application of MRM techniques to investigate the phenylpropanoid pathway proteins in plants [28,29], polyphenol oxidases in loquat fruit [30] and monodehydro-ascorbate reductase, which is a key enzyme in the redox and antioxidant system of apple fruit during ripening [31].…”