Speaker
Description
Short open reading frame (sORF) encoded proteins fulfil important roles in many cellular processes. In the methanoarchaeon Methanosarcina mazei, numerous small proteins have previously been identified under different nitrogen availabilities, however, few have been functionally characterised. Consequently, a detailed analysis of small proteins translated under a range of growth conditions may reveal interesting candidates for future downstream analyses.
We investigated the small proteome of M. mazei under five growth conditions (sufficient nitrogen, insufficient nitrogen, viral stress, heat stress, and salt stress). Here, we utilised a previously developed, top-down and bottom-up compatible, solid phase extraction methodology that enabled enrichment of the low molecular weight proteome. Subsequently, we applied both an optimised LC-FAIMS-MS top-down workflow and a standard LC-MS-based bottom-up proteomic analysis. The approach allowed for the inferred BUP identification of 234 small proteins. Additionally, top-down proteomic analysis identified 408 proteoforms for 130 protein accessions with canonical sequences less than 101 residues in length. In total, 251 small proteins could be identified via the combined approach, of which 16 were only detected by top-down analysis.
Aiming to unravel the functions of the uncharacterized small proteins, we performed sequence-based clustering with emphasis on the presence of characteristic motifs. This led to the identification of 51 small proteins containing CxxC motifs, which are potential ferredoxin-like small proteins with putative iron-sulfur (Fe-S) cluster binding-sites, or zinc-binding proteins.
Overall, the comprehensive analysis of the M. mazei small proteome under various growth conditions, using a combination of top-down and bottom-up proteomic approaches, in addition to sequence-based analyses, represents a key step in systematically uncovering the functions of small proteins in M. mazei.
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