Towards the characterization of the hidden world of small proteins in Staphylococcus aureus, a proteogenomics approach

Abstract

Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.

An analysis of proteogenomics and how and when transcriptome-informed reduction of protein databases can enhance eukaryotic proteomics

“…While this is challenging for eukaryotes due to their larger genome size, efficient approaches were developed to better characterize the coding potential of prokaryotic genomes [ 1 , 2 ]. More recently, proteogenomics has been increasingly employed to discover small proteins, since short open reading frames typically are difficult to predict by conventional genome annotation tools [ 3 , 4 ]. Proteogenomic applications were also extended to the study of gene expression regulation at transcript and protein levels or to the identification of cancer-specific protein variants [ 5 , 6 ].…”

smORFer: a modular algorithm to detect small ORFs in prokaryotes

“…We also considered recently published mass spectrometry data for S. aureus ( 41 ) and detected smORFs with SALT & Pepper pipeline ( https://gitlab.com/s.fuchs/pepper ) ( 41 ). The pipeline uses a minimum of one unique peptide larger than 6 amino acids detected in at least two biological replicates and with a minimum score of 40 for unmodified and modified peptides, a minimum delta score of 6 for unmodified peptides and 17 for modified peptides, and a fixed false discovery rate (FDR) of 0.0001 for peptides and 0.01 for proteins ( 41 ). The SALT & Pepper pipeline comprises genomic prediction of smORFs and mass spectrometry verification and detected in total 176 unique small proteins with a length of up to 100 amino acids.…”

“…The Ribo-Seq data for S. aureus Newman were uploaded in GEO under accession number GSE150601. Mass spectrometry data for S. aureus are from ( 41 ) and for E. coli from ( 42 ).…”

Peptidome: Chaos or Inevitability

“…In addition, the translation of thousands of small open reading frames (smORFs; <100 codons) located on long non-coding RNAs (lncRNAs) or mRNAs was confirmed experimentally and, therefore, is another source of peptides in cellular and secreted peptidomes [ 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. However, the abundance of these groups of peptides, their half-life and degradation mechanisms are still poorly understood [ 29 , 30 , 31 , 32 ].…”