TABLE 1.

A selection of small protein discovery studies for bacteria and archaea using shotgun (bottom-up) proteomics and top-down approaches without proteolytic digest^a

Organism(s)	Taxonomy	Approach	Notes	Sample	Reference(s)
Mycoplasma pneumoniae	Bacteria	Shotgun	Term proteogenomics introduced; search six-frame translated genome	Whole cell lysate	56
Mycoplasma mobile	Bacteria	Shotgun	Used proteomics data in initial genome annotation of an organism	Whole cell lysate	126
Shewanella oneidensis	Bacteria	Shotgun	MS-based proteomics to improve genome annotation, used PTM data, studied several conditions	Whole cell lysate; subcellular fractionation	127
Methanosarcina acetivorans	Archaea	Top down	Top-down approach identified five unannotated small proteins (40–76 aa)	Whole cell lysate	19
Staphylococcus aureus	Bacteria	Shotgun	Effort to analyze the entire expressed proteome, combining different conditions and proteomics approaches	Subcellular fractionation	128
Yersinia pestis	Bacteria	Shotgun	Required 2 peptides to identify a novel protein	Subcellular fractionation	57
Mycobacterium tuberculosis complex	Bacteria	Shotgun	Custom database approach that merges information from different strains	Various	129
46 species (bacteria and archaea)	Bacteria, Archaea	Shotgun	Large proteogenomic study; use of stringent PSM level FDR advocated	Various	30
Escherichia coli	Bacteria	Shotgun	High FDR among peptides implying novel proteins; trypsin + Lys-C	Whole cell lysate	31
Helicobacter pylori	Bacteria	Shotgun	Required 2 peptides to identify a novel protein; also used size exclusion chromatography	Whole cell lysate	130
57 bacterial species	Bacteria	Shotgun	Large proteogenomics study on N-terminal methionine excision and PTM (N-terminal acetylation)	Various	131
Saccharopolyspora erythrea	Bacteria	Shotgun	Reannotated genome of organism with high GC content (transcriptomics, shotgun proteomics)	Whole cell lysate	58
Bradyrhizobium japonicum	Bacteria	Shotgun	Custom databases to find longer (84)/shorter proteoforms (132) (integration with dRNA-seq data)	Whole cell lysate	84, 132
Synechococcus sp	Archaea	Shotgun	Proteogenomic study of model cyanobacterium (8 conditions); global profiling for PTMs (1% PSM FDR)	Whole cell lysate	133
Roseobacter denitrificans	Bacteria	Shotgun	N terminomic combined with six-frame translation database to validate/correct N termini (+ alternative proteases; Glu-C, chymotrypsin)	Whole cell lysate	27
Mycoplasma pneumoniae	Bacteria	Shotgun	Integrated anaylsis to re-annotate genome (>300 transcriptome, >70 proteome datasets); evidence for internal starts, new small proteins	Various	103
Pseudomonas stutzeri	Bacteria	Top down (MALDI)	Small transmembrane subunit of cbb3 oxidase	Purified protein complex	23
Metagenomic study of grassland soil	Bacteria, archaea	Shotgun	Metagenome-assembled genomes as basis for meta-proteomics (custom database); integrate metabolomics; beyond culturable strains	Soil extract	61
Listeria monocytogenes	Bacteria	Shotgun	N-terminal enrichment (COFRADIC approach) (26); 2nd study (92) used spectral libraries and combined DDA and DIA	Whole cell lysate	26, 29, 92
Xanthomonas euvesicatoria	Bacteria	Shotgun	Reannotation of a plant pathogen with Shotgun data; confirmed expression of 5 novel proteins with Immunoblot (c-Myc tag)	Whole cell lysate	134
Bartonella henselae	Bacteria	Shotgun	Broadly applicable proteogenomic approach, custom databases validated 107/138 peptides with PRM (97)	Subcellular fractionation	81, 97, 141
Methylobacterium extorquens	Bacteria	Shotgun	N terminome study of a strain used for microbial rehabilitation and degradation of industrial pollutants	Whole cell lysate	28
Escherichia coli	Bacteria	Top down (MALDI)	Small transmembrane subunit of bd oxidase	Purified protein complex	43
Bacillus subtilis	Bacteria	Shotgun	Explored small protein enrichment strategies, different proteases, database searches; validation by PRM and spectral matching	Small protein enrichment	12
Salmonella Typhimurium, Deinococcus radiodurans	Bacteria	Shotgun	Broadly applicable custom peptide DB; integrated Ribo-seq data and peptide fragmentation prediction	Whole cell lysates	88
Salmonella Typhimurium	Bacteria	Shotgun	Integrated small protein prediction with Ribo-seq, shotgun, and other OMICS data; elucidated function of novel small proteins	Various	104
Prokaryotes from human microbiota, Bacteroides thetaiotaomicron	Bacteria, archaea	Shotgun	Prediction of ∼4500 small protein families <50 aa; experimental evidence for selected examples (meta-transcriptomics/proteomics) Identified several small proteins in Bacteroides thetaiotaomicron	Various	1
Intestinal microbiota model system	Bacteria	Shotgun	Extended custom iPtgxDB to multispecies model (8 strains); meta-transcriptomics + meta-proteomics; spectral matching	Small protein enrichment	60
Thermosynechococcus elongatus	Bacteria	Top down (MALDI)	Small transmembrane subunit of photosystem II	Purified protein complex	44
Staphylococcus aureus	Bacteria	Shotgun	Broadly applicable approach; integrated shotgun and Ribo-seq data; automated evaluation of MS/MS spectra quality	Cytoplasmic extract	87
Methanosarcina mazei	Archaea	Shotgun/top down	Characterization of 36 proteoforms mapping to 12 small proteins with top down (2D-LC-MS)	Small protein enrichment	89
Methanosarcina mazei	Archaea	Shotgun	Multiprotease approach (SDS-PAGE)	Small protein enrichment	35

^aWe apologize to authors of the many important studies we could not reference due to space restrictions. Preference was given to more recent studies, many of which have used higher accuracy MS instruments and small protein enrichment strategies, carried out validation of novel small proteins (e.g., by PRM or spectral matching), and put a larger emphasis on integration of RNA-seq, Ribo-seq or computational small protein prediction algorithms. Proteogenomic studies prior to 2014 are more thoroughly listed by Kucharova and Wiker (125).