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Small protein enrichment improves proteomics detection of sORF encoded peptides.

ABSTRACT: With the rapid growth in the number of sequenced genomes, genome annotation efforts became almost exclusively reliant on automated pipelines. Despite their unquestionable utility, these methods have been shown to underestimate the true complexity of the studied genomes, with small open reading frames (sORFs; ORFs shorter than 300 nucleotides) and, in consequence, their protein products (sORF encoded polypeptides or SEPs) being the primary example of a poorly annotated and highly underexplored class of genomic elements. With the advent of advanced translatomics such as ribosome profiling, reannotation efforts have progressed a great deal in providing translation evidence for numerous, previously unannotated sORFs. However, proteomics validation of these riboproteogenomics discoveries remains challenging due to their short length and often highly variable physiochemical properties. In this work we evaluate and compare tailored, yet easily adaptable, protein extraction methodologies for their efficacy in facilitating the extraction and concomitantly proteomics detection of SEPs expressed in the prokaryotic model organism Salmonella Typhimurium. An optimized protocol for the enrichment and efficient detection of SEPs making use of the of amphipathic polymer amphipol A8-35 relying on the differential peptide versus protein solubility is suggested and compared with global extraction methods making use of chaotropic agents. Given the versatile biological functions the SEPs have been shown to exert, this work provides an accessible protocol for proteomics exploration of this fascinating class of small proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Typhimurium Str. Sl1344

SUBMITTER: Igor Fijalkowski

LAB HEAD: Petra Van Damme

PROVIDER: PXD025297 | Pride | 2021-10-07

REPOSITORIES: Pride

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Project description:Many small open reading frames (smORFs) embedded in lncRNA transcripts have been shown to encode biologically functional polypeptides (smORFs-encoded polypeptides, SEPs) in different organisms. Despite significant advances in genomics, bioinformatics and proteomics that largely enabled the discovery of novel SEPs, their identification across different biological samples is still hampered by their poor predictability, diminutive size and low relative abundance. Here, we take advantage of NONCODE, a repository containing the most complete collection and annotation of lncRNA transcripts from different species, to build a novel database that attempts to maximize a collection of SEPs from human and mouse lncRNA transcripts. In order to further improve SEP discovery, we implemented two effective and complementary polypeptide enrichment strategies, 30 kDa MWCO filter and C8 SPE column. These combined strategies enabled us to discover 357 and 409 SEPs from, respectively, 8 human cell lines, and 3 mouse cell lines and 8 mouse tissues. Importantly, nineteen of the identified SEPs were then verified through in-vitro expression, immunoblotting, parallel reaction monitoring (PRM) and synthetic peptides. Subsequent bioinformatic analysis revealed that some of the physical and chemical properties of these novel SEPs, including amino acid composition and codon usage, are different from those commonly found in canonical proteins. Intriguingly, nearly 65% of the identified SEPs were found to be initiated with non-AUG start codons. Overall, the strategy presented in this study encompasses an efficient workflow that enabled us to identify 766 novel SEPs across multiple cell lines and tissues, which probably represents the largest number of SEPs detected by mass spectrometry reported to date. These novel SEPs might not only provide new clues for the annotation of noncoding elements in the genome but can also serve as a valuable resource for the functional characterization of individual SEPs.

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Small protein enrichment improves proteomics detection of sORF encoded peptides.

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