Exploration of Alternative Proteases in Shotgun Proteomics to Increase Proteome and Splice Junction Coverage in Plants

Alternative proteases can be used to mitigate the issues associated with tryptic digestion in shotgun proteomics and increase coverage of the proteome. Existing programs to aid in digest scheme selection focus on a proteomic context only. This work aims to develop a tool to extend in silico digest scheme analysis to a proteogenomic context and evaluate its performance by generating proteomic data using optimised protocols for alternative protease digests in vitro. I present ProtView, a versatile in silico digest evaluation tool that maps in silico-digested peptides to both protein and genome references, allowing potential observable portions of the proteome, transcriptome, and genome to be identified. ProtView was used to select proteases to digest Barley and Arabidopsis tissue, which were then digested using optimised protocols for the selected proteases. In Barley, the transcriptomic database BaRTv2.18 (Coulter et al., 2022) was queried using proteomic data to identify splice-junction covering peptides. In Arabidopsis, a simplified version of a time-series cold acclimation experiment was performed, to explore proteomic evidence of the cold response. In turn, these datasets were used to evaluate ProtView. Proteomic evidence was found for an upper limit of 13,924 genes, 62,900 isoforms, and 16,172 unique exon-exon junctions from the translated BaRTv2.18 database. This is an additional 3,433 genes, 11,329 isoforms, and 4,782 unique exon-exon junctions to those potentially identifiable by tryptic digestion alone. In Arabidopsis an upper limit of 10,433 genes, 19,051 isoforms and 9,688 unique exon junctions were identified, increasing on possible tryptic identifications by 1,837 genes, 3,332 isoforms and 2,559 unique exon junctions. These results support the use of ProtView to aid in digest scheme selection in both a proteomic and proteogenomic context and highlight the merits of using multiple proteases in parallel combination. Alternative splicing patterns between timepoints were also found for 357 proteins in Arabidopsis.