Is Peptide Yield enhanced by repeated Phospho-enrichments in Brassicas?

Is Peptide Yield enhanced by repeated Phospho-enrichments in Brassicas?

Life Sciences

Brassica oleracea is a common family of crop plants susceptible to disease. In this work the research groups' Mass Spectrometry-based Phosphoproteomics method was improved by increasing TiO2 enrichment steps. This enabled the research group to identify key phosphoproteins involved in immune signal transduction.

Sean Connors

2021

Brassica oleracea is a common family of crop plants including Cabbage, Broccoli, Cauliflower, Kale and Brussels sprout. Brassicas are susceptible to a host of pests and disease such as Black rot. Immune signal pathways, consisting of essential phosphorylation events, remain poorly studied and unclear in many crop plants such as Brassica oleracea.

To identify the most phosphoproteins, high-throughput mass spectrometry-based phosphoproteomics was used. The research group’s standard protocol involves a single TiO2 enrichment step, but still misses some essential phosphoproteins. To investigate whether increasing the number of enrichments improves the phosphoprotein identification, three enrichment steps were used and over 1000 phosphoproteins from 1370 unique peptides were identified. The 3 phosopho-peptide enrichment steps generally increased the peptide yield by 25% compared to the standard 1 enrichment. The difference in physiochemical properties between samples was caused by a higher abundance of more hydrophilic and highly phosphorylated serine peptides in the 1st enrichment.

This observed enhancement in phosphopeptide yield will enable a significant increase in phosphoprotein identification and demonstrate more novel phosphorylation events related to immune pathways. Characterising these mechanisms will provide direct experimental evidence for resistance genes and will play an important role in annotating these genes. This annotation can be used to breed disease resistance crops and increase food security.