The Power of Three in Cannabis Shotgun Proteomics: Proteases, Databases and Search Engines

Cannabis research has taken off since the relaxation of legislation, yet proteomics is still lagging. In 2019, we published three proteomics methods aimed at optimizing protein extraction, protein digestion for bottom-up and middle-down proteomics, as well as the analysis of intact proteins for top-down proteomics. The database of Cannabis sativa proteins used in these studies was retrieved from UniProt, the reference repositories for proteins, which is incomplete and therefore underrepresents the genetic diversity of this non-model species. In this fourth study, we remedy this shortcoming by searching larger databases from various sources. We also compare two search engines, the oldest, SEQUEST, and the most popular, Mascot. This shotgun proteomics experiment also utilizes the power of parallel digestions with orthogonal proteases of increasing selectivity, namely chymotrypsin, trypsin/Lys-C and Asp-N. Our results show that the larger the database the greater the list of accessions identified but the longer the duration of the search. Using orthogonal proteases and different search algorithms increases the total number of proteins identified, most of them common despite differing proteases and algorithms, but many of them unique as well.

Keywords: Asp-N; LC-MS; Mascot; SEQUEST; bottom-up and middle-down proteomics; cannabis sativa; chymotrypsin; missed cleavages; post-translational modification; trypsin/Lys-C.