panorama0000ProteomicsJuergen BartelBacillus Subtilishttps://panoramaweb.org/SmallProteinsBSubtilis.urljuergen.bartel@uni-greifswald.deUniversity of Greifswald, Center of Functional Genomics of Microbes, Institute of Microbiology, Department of Microbial ProteomicsPanoramaPublicSmall open reading frame encoded proteins (SEPs) gained increasing interest during the last few years because of their broad range of important functions in both prokaryotes and eukaryotes. In bacteria, signaling, virulence, and regulation of enzyme activities have been associated with SEPs. Nonetheless, the number of SEPs detected in large-scale proteome studies is often low as classical methods are biased toward the identification of larger proteins. Here, we present a workflow that allows enhanced identification of small proteins compared to traditional protocols. For this aim, the steps of small protein enrichment, proteolytic digest, and database search were reviewed and adjusted to the special requirement of SEPs. Enrichment by the use of small-pore-sized solid-phase material increased the number of identified SEPs by a factor of 2, and utilization of alternative proteases to trypsin reduced the spectral counts for larger proteins. The application of the optimized protocol allowed the detection of 210 already annotated proteins up to 100 amino acids (aa) length, including 16 proteins below 51 aa in the Gram-positive model organism <i>Bacillus subtilis</i>. Moreover, 12% of all identified proteins were up to 100 aa, which is a significantly larger fraction than that reported in studies involving traditional proteomics workflows. Finally, the application of an integrated proteogenomics search database and extensive subsequent validation resulted in the confident identification of three novel, not yet annotated, SEPs, which are 21, 26, and 42 aa long.Optimized Proteomics Workflow for the Detection of Small Proteins.Bartel Jürgen J, Varadarajan Adithi R AR, Varadarajan Adithi R AR, Sura Thomas T, Ahrens Christian H CH, Maaß Sandra S, Becher Dörte DFBgn0003149, scale tissue, HSN1E, Aminosaeure, Mbp1, Amino acid, protein, infectivity, Social Controls, Mendosicutes, Techniques, Para, Method, Bacillus subtilis subsp. natto, Work Flow, protein aggregate, CG5939, myd, animal, Formal Social Controls, viral infection, increased, me75, Eubacteria, pore, amino acids, Metabacteria, Bacillus mesentericus, plant peltate hair, hypoplasia, Mbp-1, procedures, Eucarya, D17Mit170, T1, eucaryotes, Bacteria Woese et al. 2024, Social, g, Bacteria <bacteria>, Methodological Studies, Prokaryotae, signaling process, Procaryotae, CXXC finger protein 9, single organism signaling, Archaebacteria, wide/broad, gyltl1b-b, DmelCG5939, Aminokarbonsaeure, Procedure, Tl3, Tl2, alpha-amino carboxylic acids, organism, Workflows, prm, MDDGA6, mKIAA0609, Pathogenicity, prokaryotes, l(3)S010605, KIAA0609, fg, Amino Acid, Acid, ADCADN, gyltl1b, whole organism, UNQ203/PRO229, Amino acids, 0106/05, DNA (cytosine-5-)-methyltransferase 1, PM/mPM, mdc1d, Eukarya, Control, expanded, Methodological, Controls, study protocol, Methodological Study, beta Trypsin, Bacillus subtilis (natto), MDC1D, wide, enr, enlarged, Manuscripts, Koerper, Biocatalyst, Acids, Bacillus globigii, Regulation, Proteomes, Work Flows, Prokaryota, accessory, Regulations, big, biological signaling, multi-cellular organism, Procedures, Peptidomics, Biocatalysts, peltate hair, number, Aminocarbonsaeure, Eukaryotes, Gene, long., eubacteria, broad, alpha-amino acid, protein-containing complex, supernumerary, froggy, Bacillus uniflagellatus, Gyltl1a, large, Aim, method, AIM, reduced, mPM, method used in an experiment, Gene Products, Studies, Low, tiny, Bacteriobiota, Technique, DNMT1, DNMT1_HUMAN, MDDGB6, Bacillus subtilis8, beta-Trypsin, LARGE, Monera, Bacteria (ex Cavalier-Smith 1987), Study, BPFD#36, API6, Enzyme, Vibrio subtilis, great, Natto Bacteria, fungi, species, Archaebiota, DNA MTase HsaI, small, DNA (cytosine-5)-methyltransferase 1, bacteria, Eukaryote, cou, Formal Social Control, protein complex, body, Proteins, DNMT, total expressed protein, whole body, Eukaryotas, alpha-amino acids, MCMT, Bacillus subtilis/Bacillus globigii, eukaryotes, Lr, native protein, Social Control, DNA methyltransferase HsaI, Protein, techniques, scales, Data Base, CXXC9, Bacillus subtilis var. natto, Bacillus natto, PRSS, scale, CT-2, underdeveloped, not Bacteria Haeckel 1894, increased number, archaea, Tripcellim, pore-forming toxin activity, Amino, CXXC-type zinc finger protein 9, signalling, Protein Gene Products, plan specification, Gene Proteins, present in greater numbers in organism, Trypure, Eukaryotae, signalling process, l(3)10631, prokaryote, cardinality, Bra, virulence, regulation, Eucaryotae, CLEC2C, pm, PM, methodology, m.HsaI, channel-forming toxin activitysmall, Protein Gene Products, Gene Proteins, Workflows, reduced, underdeveloped, Peptidomics, Protein., Protein, Gene Products, Proteins, hypoplasia, Gene, tiny, Work Flow, Work Flowssmall, Protein Gene Products, Gene Proteins, Workflows, reduced, underdeveloped, Peptidomics, Protein., Protein, Gene Products, Proteins, hypoplasia, Gene, tiny, Work Flow, Work Flowsscale tissue, HSN1E, Proteolytic Enzyme, Aminosaeure, Mbp1, Amino acid, protein, Reading Frame, infectivity, Social Controls, Mendosicutes, Esteroproteases, Techniques, Hydrolase, Method, Bacillus subtilis subsp. natto, ORFs, Proteases, Work Flow, protein aggregate, Unassigned, myd, animal, Unassigned Reading Frame, Formal Social Controls, Unidentified Reading Frames, viral infection, me75, Eubacteria, pore, amino acids, Metabacteria, Bacillus mesentericus, plant peltate hair, Open, hypoplasia, Mbp-1, procedures, Eucarya, Proteolytic, D17Mit170, T1, Proteinases, eucaryotes, Bacteria Woese et al. 2024, Social, Protein Coding, g, Bacteria <bacteria>, Methodological Studies, Prokaryotae, Procaryotae, Protein Coding Regions, CXXC finger protein 9, single organism signaling, Archaebacteria, Proteinase, gyltl1b-b, statistics and numerical data, Aminokarbonsaeure, Data Base., Procedure, Tl3, Tl2, alpha-amino carboxylic acids, organism, Workflows, MDDGA6, mKIAA0609, Pathogenicity, numerical data, prokaryotes, Region, KIAA0609, fg, ORF, Amino Acid, Acid, ADCADN, gyltl1b, whole organism, UNQ203/PRO229, Amino acids, DNA (cytosine-5-)-methyltransferase 1, mdc1d, Eukarya, Control, expanded, Methodological, Controls, study protocol, Methodological Study, beta Trypsin, Bacillus subtilis (natto), MDC1D, enr, enlarged, Koerper, Biocatalyst, Acids, Bacillus globigii, Regulation, Proteomes, Work Flows, Prokaryota, Proteolytic Enzymes, Regulations, big, multi-cellular organism, Procedures, Open Reading Frame, Peptidomics, Biocatalysts, peltate hair, Aminocarbonsaeure, Eukaryotes, Gene, Protease, eubacteria, alpha-amino acid, protein-containing complex, froggy, Bacillus uniflagellatus, Gyltl1a, Unidentified Reading, large, Aim, method, AIM, reduced, utilization, method used in an experiment, Unassigned Reading Frames, Gene Products, Studies, Low, Bacteriobiota, tiny, Peptide Hydrolase, Technique, Frame, DNMT1, sORF, DNMT1_HUMAN, MDDGB6, Bacillus subtilis8, Coding Region, beta-Trypsin, LARGE, Monera, Unidentified Reading Frame, Bacteria (ex Cavalier-Smith 1987), Study, BPFD#36, API6, Enzyme, Vibrio subtilis, great, Natto Bacteria, fungi, species, Archaebiota, DNA MTase HsaI, small, DNA (cytosine-5)-methyltransferase 1, Protein Coding Region, bacteria, use, Eukaryote, cou, Formal Social Control, protein complex, body, Proteins, DNMT, total expressed protein, whole body, Eukaryotas, alpha-amino acids, MCMT, Bacillus subtilis/Bacillus globigii, Peptide, eukaryotes, Lr, native protein, Social Control, DNA methyltransferase HsaI, Protein, Peptidases, Small Open Reading Frames, techniques, scales, Data Base, CXXC9, Bacillus subtilis var. natto, Bacillus natto, PRSS, scale, CT-2, underdeveloped, not Bacteria Haeckel 1894, archaea, Tripcellim, pore-forming toxin activity, Amino, CXXC-type zinc finger protein 9, Unidentified, Protein Gene Products, plan specification, Gene Proteins, Trypure, Peptidase, Eukaryotae, signalling process, prokaryote, Bra, Small Open Reading Frame, virulence, regulation, Eucaryotae, CLEC2C, methodology, m.HsaI, channel-forming toxin activity0falseAn Optimised Proteomics Workflow for the Detection of Small Proteins.Small open reading frame encoded proteins (SEPs) gained increasing interest during the last years due to their broad range of important functions in both, prokaryotes and eukaryotes. In bacteria, signalling, virulence or regulation of enzyme activities have been associated with SEPs. Nonetheless, the number of SEPs detected in large-scale proteome studies is often low as classical methods are biased towards the identification of larger proteins. In the accompanying manuscript, we present a workflow that allows enhanced identification of small proteins compared to traditional protocols. For this aim, the steps of small protein enrichment, proteolytic digest and database search were reviewed and adjusted to the special requirement of SEPs. Enrichment by the use of small-pore-sized solid-phase material increased the number of identified SEPs by a factor of two and the utilisation of alternative proteases to trypsin reduced spectral counts for larger proteins. The application of the optimised protocol allowed the detection of 210 already annotated proteins up to 100 amino acids length, including 16 proteins below 51 amino acids in the Gram-positive model organism Bacillus subtilis. Moreover, 12% of all identified proteins were up to 100 amino acids which is a significant larger fraction than reported in studies involving traditional proteomics workflows. Finally, an integrated proteogenomics search database was applied to identify potentially novel SEPs. This submission contains PRM data generated in order to verify the identification of novel very small proteins in Bacillus subtilis. Three SEPs, which are 21, 26 and 42 amino acids long, respectively were successfully verified.Tue Sep 08 00:00:00 BST 2020PXD020514142332812434