detection via genomic tagging and immunodetection of candidate sORFs Vanorsdel et al. 2020) suggest that sORFs may even be counted in hundreds but experimental confirmation of these data however is only limited with few high-throughput methods reported (Miravet‐Verde et al. Algorithms used for prokaryotic sORF delineation without a priori knowledge (Hemm et al. 2019), the latter omics approaches jointly referred to as riboproteogenomics (Ndah et al. 2021) and proteomics efforts (Impens et al. 2019), transcriptomics, translatomics (Venter et al. Yet, numerous novel bacterial sORFs and SEPs have recently been discovered in bacteria using phylogenetic analyses (Sberro et al. While current annotation pipelines largely rely on sequence composition features instead of length thresholds to distinguish coding from noncoding sequences, short proteins still remain underrepresented (Warren et al. 2008, Richardson and Watson 2013, Miravet‐Verde et al. In the past, predicted ORFs were frequently penalized based on length using arbitrary cutoffs (like 100 codons) and sequence overlap with other ORFs (Dinger et al. These properties situate complete SEP identification as an important milestone on the way to better our understanding of bacterial systems biology. A pivotal role of SEPs in bacterial biology is also supported by the fact that sORFs have been found to be the most frequently essential genomic elements of the Mycoplasma pneumoniae genome (Lluch‐Senar et al. 2014, Duval and Cossart 2017, Fijalkowska et al. In bacteria, SEPs have been associated with a plethora of cellular functions, including glucose uptake, antibiotic resistance and peptidoglycan synthesis to name a few (Storz et al. Besides regulatory sORFs, many sORFs have been shown to give rise to functionally relevant protein products (Park et al. Thus far, existence of actively translated sORFs has been detected in a wide variety of organisms. One particularly understudied class of genomic elements is represented by small open reading frames (sORFs), generally considered as ORFs encoding proteins (equal or) smaller than 100 amino acids in length and herein referred to as sORF-encoded polypeptides or SEPs (Miravet‐Verde et al. Despite their utility, a growing body of evidence suggests that these methods pose a danger of propagating biases present in current annotations and may vastly underestimate the complexity of bacterial genomic architecture (Crappé et al. With rapidly growing number of sequenced genomes, manual gene annotation had to give way to automated gene prediction algorithms. Salmonella Typhimurium, sORF-encoded polypeptides (SEPs), sORF, riboproteogenomics, proteomics, in silico proteomics Introduction Taken together, our study pinpoints current limitations in proteomics-based detection of novel small proteins currently missing from bacterial genome annotations. Typhimurium across growth phases and infection-relevant conditions. This integrative approach is used to provide a data-driven census of small proteins expressed by S. Typhimurium), a model human pathogen, across a panel of growth conditions is presented and used in support of our in silico SEP detectability analysis. Moreover, a large-scale proteomics and translatomics compendium of proteins produced by Salmonella Typhimurium ( S. A panel of physiochemical properties alongside recently developed mass spectrometry detectability metrics was interrogated to provide a comprehensive evidence-based assessment of sORF-encoded polypeptide (SEP) detectability. In this study, we provide a large-scale riboproteogenomics investigation of the challenging nature of proteomic detection of such small proteins as informed by conditional translation data. Despite the mounting genomic evidence of their robust expression, relatively little progress has been made in their mass spectrometry-based detection and various blanket statements have been used to explain this observed discrepancy. Genomic studies of bacteria have long pointed toward widespread prevalence of small open reading frames (sORFs) encoding for short proteins, <100 amino acids in length.
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