Significant number of small proteins with less than 50 amino acids remain uncharacterized in different microbial species

Small proteins have attracted significant research interest recently principally as it was a neglected class of proteins in the early proteomics era. Usually running far ahead of larger proteins during gel electrophoresis, small proteins typified by having fewer than 50 amino acid residues are not commonly analyzed and thus neglected. In addition, some mass spectrometry methods such as matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) could not ionize small proteins efficiently, or their flight time could not be timed precisely to afford routine detection of such proteins. On the other hand, while small proteins would theoretically be detected by liquid chromatography tandem mass spectrometry (LC-MS/MS), short retention time presents challenges to their facile analytical detection. Hence, the experimental picture of small proteins cast the light that they could not be efficiently detected and analyzed, which meant that they may not be well characterised. Such a postulation is validated by the present work that sought to understand the types of proteins that constitute small proteins in different microbial species. Results reveal that significant number of small proteins in different microbial species that span bacteria, archaea and eukaryotes are not characterised. No function or name could be annotated for such proteins, which suggests that understanding of their existence remain at the level of gene sequence profiled by automatic gene finder during whole-genome sequencing and assembly. This then opens up a vast vista for the detection, isolation, purification, and analysis of such uncharacterised proteins. Knowledge of their gene sequence offers the first step as it opens the way for their heterologous expression in <i>Escherichia coli</i>, which provides sufficient protein material for isolation, purification, and characterisation in downstream biochemical assays. Overall, knowledge of small proteins at the level of biochemical function, and binding partners remain poor due in large part of their uncharacterised status in different microbial species. Future work could build on our understanding of the genetic repertoire of microbial species to proactively express, through heterologous means, small proteins for biochemical characterisation. Attached with this preprint is a list of microorganisms with significant number of uncharacterised small proteins.