Small Protein Enrichment Improves Proteomics Detection of sORF Encoded Polypeptides

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 typically considered 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 the extraction and concomitantly proteomics detection of SEPs expressed in the prokaryotic model pathogen Salmonella typhimurium (S. typhimurium). Further, an optimized protocol for the enrichment and efficient detection of SEPs making use of the of amphipathic polymer amphipol A8-35 and relying on differential peptide vs. protein solubility was developed and compared with global extraction methods making use of chaotropic agents. Given the versatile biological functions SEPs have been shown to exert, this work provides an accessible protocol for proteomics exploration of this fascinating class of small proteins.

Shift in vacuolar to cytosolic regime of infectingSalmonellafrom a dual proteome perspective

“…Typhimurium cultures and triplicate shotgun proteome analysis, typically a 2-fold increase in the number of S . Typhimurium proteins identified and quantified (48) (around ~ 1,500 to 1,600) are found using similar MS instrumentation in DDA mode (30).…”

Exposing the Small Protein Load of Bacterial Life

“…With documented bacterial SEP functions falling within diverse categories of basic and essential bacterial physiology [23][24][25][26][27][28] as well as infection biology [29][30][31][32], the need for more large-scale validation and functional characterization efforts is high. In this context, it is noteworthy that difficulties in biochemical detection and therefore validation of SEPs are known and have been extensively documented [33][34][35], but that also recent bacterial SEP validation studies fail to fully address many of the challenges in small protein detection [35], such as their proposed low expression or low stability [36]. Nonetheless, as expression detection is a prerequisite for functional investigations, further improvement in SEP detection might turn out to be of great value to expand our current understanding of bacterial (infection) biology.…”

“…The trend observed showed a clear correlation between the number of theoretical detectable peptides and the length of the protein from which the theoretical peptide descends, a conclusion logically linking SEP detection difficulties to size. Protocols aiming at high-molecular-weight protein depletion [85] or low-molecular-weight protein enrichment [34] have been proposed to increase peptide identification rate and coverage of the limited theoretically identifiable peptide arsenal originating from SEPs [86], but these technologies forego the quantitative aspect of proteomics data and are therefore not generally applicable. Also the use of a more diverse set of MS-sequencing proteases for proteome digestion might benefit SEP identification through increased sequence coverage as was done during a recent proteogenomic study of the Y. pestis genome [48].…”

Microproteins—Discovery, structure, and function

“…Therefore, the first critical step of any sORF proteomic experiment is to achieve proteome extraction in the absence of proteolysis of canonical proteins (e.g., via boiling in acidic solution or application of protease inhibitors [84]) to minimize sample complexity, followed by or concomitant with enrichment of the small proteome and exclusion of large proteins. Small protein enrichment can be achieved via multiple chemical and biophysical methods, such as solid phase extraction, peptide gels, GELFrEE resolution, and organic solvent or surfactant extraction [88][89][90][91][92]. When they have been compared head-to-head, these methods have typically been shown to offer comparable numbers, but non-overlapping sets, of microproteins detected [88][89][90][91].…”