Guided tour through the small protein landscape of Methanosarcina mazei using proteomics and biochemical approaches.

Abstract

Small open reading frame (small ORF) 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, with only few being subject to functional characterization. Consequently, a detailed expression analysis of small proteins translated under other stress conditions, in conjunction with conservation and sequence-based analyses, may reveal interesting candidates for future downstream analyses. Here, we investigated the small proteome of M. mazei under five growth conditions. By enriching the low molecular weight proteome and combining top-down and bottom-up proteomic analysis, a total of 234 small proteins were validated on protein level, of which 130 were found in top-down proteomics analysis, which were associated with 408 proteoforms. Aiming to unravel functions of the large number of small proteins, we performed sequence-based clustering with emphasis on the presence of characteristic motifs. Thereby, ferredoxin-like small proteins with putative iron-sulphur (Fe-S) cluster binding-sites, as well as possible zinc-binding proteins, both with distinct cysteine motifs, were identified. We further analysed heterologously expressed representatives of the ferredoxin-like and putative zinc-binding small proteins, confirming the zinc-binding capability of two small proteins of the latter group via inductively coupled plasma-mass spectrometry. Overall, the detailed analysis of the M. mazei small proteome under different growth conditions, using various proteomics approaches, as well as sequence-based analyses and biochemical approaches targeting specific protein candidates, represents a key step in systematically uncovering the functions of small proteins in M. mazei.