Background
Decoding the protein repertoire of field pea (Pisum sativum) samples across various developmental stages can greatly increase our understanding of the molecular mechanisms involved in protein accumulation, yield and nutritional quality. However, the proteome-level transition between different developmental stages during the maturation periods remains unknown. In this international collaborative project between CSIRO and National Research Council (NRC) Canada, we performed a proteogenomics experiment on 10 pea cultivars across five developmental stages.
Methodology
Pea samples were grown by NRC investigators in Saskatchewan province in Canada. Ten pea cultivars were grown and samples were collected across five developmental stages:11, 14, 19, 24 and 29 days, post anthesis (DPA), respectively, in 3 biological replicates. Proteomics data were acquired using sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS)- based approach on a TripleTOF 6600 MS instrument (ABSCIEX, USA). The resulting data files were processed using an in-house constructed database and DIA-NN approach. Bioinformatics and functional analyses were performed on differentially abundant proteins between the five developmental stages and ten lines. Targeted multiple reaction monitoring (MRM) assays were developed to measure selected seed storage proteins identified in the SWATH-MS analysis across the developmental stages to monitor their accumulation pattern.
Results
The processed data files using DIA-NN software resulted in the quantitation of 23,565 peptides mapped to 6,219 proteins at a 1% false discovery rate. Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) were performed on the resulting data matrix, revealing that the samples were clustered based on the developmental stages. The two early developmental stages were closely clustered compared to the three late stages based on the measured protein abundance changes. As an example, for a selected cultivar, the comparison between stages 1 and 2 revealed the abundance changes of ~10% of detected proteins (Fold Change =2; p<0.05). Functional annotations of the altered proteins revealed the increase of catalytic activities, and storage protein accumulations were upregulated in the late developmental stages compared to the early stages. The MRM data revealed a distinct pattern of storage protein subtype accumulation across the developmental stages.
Conclusion
We envision this comprehensive study on pea proteome across five developmental stages can greatly enhance our fundamental knowledge of storage protein accumulation and proteome diversity, which can assist in reducing anti-nutritional proteins and improving nutritionally enriched protein contents either through selective breeding or genome engineering.
Keywords: Field pea, Pisum sativum, Plant protein, SWATH-MS, MRM-MS