Abstract
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.
Highlights
Bottom-up proteomics (BUP) refers to the characterization of proteins by analysis of their peptides released through proteolysis
As Middle-down proteomics (MDP) can be applied to a complex mixture of proteins, it falls under the shotgun proteomics category
In order to enable meaningful comparisons across proteases, every sample preparation step was kept rigorously identical for each sample, with the exception of the digestion steps themselves where optimum conditions were applied as recommended by the manufacturer to maximise protease efficiency
Summary
Bottom-up proteomics (BUP) refers to the characterization of proteins by analysis of their peptides released through proteolysis. We developed a top-down proteomics strategy to analyse intact proteins and discovered that cannabis proteins are predominantly methylated [3] These three proof-of-concept proteomics studies share one deficiency: the UniProt database searched for the purpose of protein identification was small (containing less than 500 entries) and greatly underestimated the rich genetic diversity of C. sativa [33]. We take this opportunity to compare two very popular search engines: SEQUEST and Mascot In this fourth experiment, we present shotgun proteomics results related to single parallel digestions using orthogonal proteases of decreasing selectivity, namely Asp-N, Trypsin/Lys-C and chymotrypsin. Our observations are presented and discussed with respect to LC-MS patterns, database search yield and duration, search algorithm comparison, protease efficiency, number of accessions, number of missed cleavages and peptide size, PTMs and gene ontology
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