In the last years, we learned a lot about bacteria and their genomes. However, their gene products are often still mysterious to us. Especially proteins of small size are mainly unrecognized and thus uncharacterized. The study “Hidden in plain sight: challenges in proteomics detection of small ORF-encoded polypeptides” in microLife aims to use proteomics approaches to detect small bacterial proteins in the pathogen Salmonella enterica. Petra Van Damme and Igor Fijalkowski explain for the #FEMSmicroBlog how this could advance the proteomics field and what these new proteins could be needed for. #FascinatingMicrobes
Small bacterial genes and proteins are out of sight
Recent years brought massive improvements in our understanding of bacterial genomes. Sequencing-based technologies helped us get insights into bacterial DNA, RNA production and its translation into mature proteins.
These newfound understandings unravelled certain classes of genes and open reading frames (ORFs) that we couldn’t discover before and were therefore understudied. Interestingly, small ORFs represented one such class of hidden genomic elements in the “genomic dark matter”. These encode small proteins, also termed short ORF encoded polypeptides or SEPs.
Small open reading frames encoding short ORF encoded polypeptides are hidden genomic elements in the genomic dark matter.
Since short genomic coding sequences are more difficult to separate from genomic noise, they are prone to false detection. Therefore, typical arbitrary cut-offs prevent the detection of small genes. Lastly, the resulting protein products are shorter than 100 amino acids in length, so that their detection is rather challenging.
Yet, a limited number of SEPs were described and functionally characterized to date. Interestingly, many of them were shown to be involved in profound and essential processes in bacteria, ranging from metabolism to antibiotic resistance.
Despite these genomic advances, one aspect of SEP characterization is clearly lagging – their proteomic detection. Finding the coding region of a new gene is a challenge on its own, but the ultimate proof of its protein-coding potential is detecting its translation product – the mature protein.
Detecting small bacterial proteins with proteomics
On a global scale, this is achieved through mass-spectrometry based proteomics. The sensitivity of mass spectrometers has increased greatly in the last decade, yet, improvements in detecting SEPs did not follow.
The study “Hidden in plain sight: challenges in proteomics detection of small ORF-encoded polypeptides” in microLife analyzes the reasons behind this hindrance. This work aims to better understand the current shortcomings of proteomics and guide further development in this field to detect SEPs.
For this, the study uses the model bacterial pathogen Salmonella enterica serovar Typhimurium. It analyzes the properties of annotated small proteins that were currently not detected by proteomics approaches. It seems that both, the number of produced peptides and their low mass-spectrometry detectability, disproportionally affect detecting SEPs compared to longer proteins.
The number of produced peptides and the low mass spectrometry detectability of SEPs affect their identification.
Ribosome profiling further gave conclusions about protein production during translation. When ribosomes produce proteins from mRNA templates, their activity can be stopped by specific antibiotics. At this moment, (initiating) ribosomes in complex with mRNA are present in the cell and can be isolated. Sequencing the enclosed mRNA fragments allowed to produce a detailed map of all ongoing protein production across the entire genome at that moment.
New small bacterial proteins are involved in many processes
The protein sequences for the novel and previously uncharacterized SEPs helped improve proteomics databases. These new entries led to new information on the Salmonella genome, which finally made the current genome annotation more accurate.
Moreover, this study analysed bacteria in various growth conditions to search for proteins produced in response to particular environmental stressors and during infection. Interestingly, some of these new SEPs are specifically expressed in infection-relevant conditions. Hence, future functional studies should focus on fully understanding the biological relevance of these newly discovered ORFs.
Reannotation of genomes based on functional information is an ongoing challenge. Yet again, It became increasingly clear that specific classes of particularly challenging genomic elements, such as small ORFs and their encoding SEPs, require specialized approaches for their characterization.
- Read the article “Hidden in plain sight: challenges in proteomics detection of small ORF-encoded polypeptides” in microLife by Fijalkowski et al. (2022).
About the authors of this blog
Petra Van Damme holds a Professorship (senior lecturer) at Ghent University. She obtained her master’s degree in Biochemistry in 2001 and obtained her PhD in Biomedical Sciences in 2008. Her doctoral research was oriented toward the field of gel-free proteomics and focused on the development of proteome-analytical methods for protein identification and characterization. Afterwards, her research focus diverged from the study of proteolysis to the study of N-terminal protein modifications and more recently, the application of riboproteogenomics strategies to map the translation (initiation) landscape in bacteria and eukaryotes. Her recent discoveries relate to the existence of N-terminal proteoforms and improving the genome annotation of bacterial pathogens such as Salmonella (in the context of infection).
Igor Fijalkowski obtained his PhD in 2016 at the Center of Medical Genetics in Antwerp, where he studied rare bone diseases and worked towards transforming the acquired knowledge into novel treatment strategies for osteoporosis. Since then, he has been a postdoctoral researcher in the Integrative Riboproteogenomics, Interactomics and Proteomics (iRIP) Lab headed by Prof. Van Damme within the UGent Laboratory of Microbiology. His postdoctoral research focuses on multi-omics integration utilizing transcriptomics, translatomics and proteomics to investigate the mechanisms of translation and search for novel genomic elements in the unexplored genomic dark matter.
About this blog section
The section #FascinatingMicrobes for the #FEMSmicroBlog explains the science behind a paper and highlights the significance and broader context of a recent finding. One of the main goals is to share the fascinating spectrum of microbes across all fields of microbiology.
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