Table 2.
Identification of S-Bacillithiolated Proteins in the Proteomes of B. subtilis, B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus Using Liquid Chromatography-MS/MS Analysis
| Accession | Protein | Function | Essential | Redox-sensing Cys | Peptide-SSB or -SSCys sequence | m/z precursor | m/z prec-134 (-malate) | Peptide neutral MW | Charge |
|---|---|---|---|---|---|---|---|---|---|
| B. subtilis | |||||||||
| O34777 | OhrR* | Organic hydroperoxide resistance repressor (control of the OhrA peroxiredoxin) | No | C15 redox sensing (cons.) | (K)LENQLC15(+BSH)FLLYASSR(E) | 684.9800 | 640.6500 | 2051.9183 | 3 |
| P80877 | MetE* | 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (methionine synthase) | No | C647,730 Zn-binding active site (cons.) | (R)FWVNPDC730(+BSH)GLK(T) | 787.8297 | 720.9900 | 1573.6449 | 2 |
| (R)VPSTEEMYNIIVDALAVC719(+BSH)PTDR(F) | 944.7584 | 900.4600 | 2831.2533 | 3 | |||||
| P42318 | YxjG* | Methionine synthase homolog | No | C346 Zn-binding active site (cons.) | (R)YVSLDQLC341(+IAM)LSPQC346(+BSH)GFASTEEGNK(L) | 981.4173 | 936.8900 | 2941.2301 | 3 |
| P37487 | PpaC* | Manganese-dependent inorganic pyrophosphatase | Yes | C158 cons. | (K)SPTC158(+BSH)TDQDVAAAK(E) | 851.8443 | 785.1000 | 1701.6740 | 2 |
| P35136 | SerA* | d-3-phosphoglycerate dehydrogenase (serine biosynthesis) | No | C410 cons. in Bacillus species | (K)ISSSESGYDNC410(+BSH)ISVK(V) | 992.9086 | 926.0500 | 1983.8026 | 2 |
| P39912 | AroA | Phospho-2-dehydro-3-deoxyheptonate aldolase/chorismate mutase (chorismate biosynthesis) | No | C126 cons. | (R)FIVGPC126(+BSH)AVESYEQVAEVAAAAK(K) | 883.4080 | 839.4800 | 2647.2022 | 3 |
| P33166 | Tuf | Elongation factor Tu | Yes | C83 in GTP-binding site 82–86 (cons.) | (R)HYAHVDC83(+BSH)PGHADYVK(N) | 527.7176 | 494.4700 | 2106.8413 | 4 |
| P21879 | GuaB | Inosine-5′-monophosphate dehydrogenase (GMP biosynthesis) | Yes | C308 active site (cons.) | (K)VGIGPGSIC308(+BSH)TTR(V) | 519.5691 | 475.0800 | 1555.6854 | 3 |
| P54170 | YphP*,# | UPF0403 protein, thioredoxin-like protein | No | C53 active site (cons.) | (K)AEGTTLVVVNSVC53(+BSH)GC55(+IAM)AAGLAR(P) | 815.3758 | 771.1400 | 2443.1056 | 3 |
| O05268 | YumC | Ferredoxin—NADP reductase 2 (FAD-dependent pyridine nucleotide-disulfide oxidoreductase) | Yes | C85 probably active site (cons.) | (K)FDQTIC85(+Cys)LEQAVESVEK(Q) | 653.3026 | n.d. | 1956.8860 | 3 |
| P18255 | ThrS | Threonine—tRNA ligase 1 | No | C338 Zn-binding; C573 not cons. | (R)LQC573(+BSH)EGLR(V) | 607.7535 | 540.8300 | 1213.4925 | 2 |
| O31678 | QueF | NADPH-dependent 7-cyano-7-deazaguanine reductase (tRNA-Queuosine biosynthesis) | No | C56 active Zn-binding site (cons.) | (K)FNC49(+IAM)PEFTSLC56(+BSH)PK(T) | 613.5820 | 569.1400 | 1837.7241 | 3 |
| B. amyloliquefaciens | |||||||||
| A7Z3U1 | MetE | 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (methionine synthase) | No | C647,730 Zn-binding active site (cons.) | (R)FWVNPDC730(+BSH)GLK(T) | 787.8297 | 721.0100 | 1573.6448 | 2 |
| (R)FWVNPDC730(+Cys)GLK(T) | 433.1948 | n.d. | 1296.5626 | 3 | |||||
| (R)VPATEEIYQIIDDALEVC719(+BSH)PTDR(F) | 962.7668 | 918.2900 | 2885.2787 | 3 | |||||
| A7ZAR2 | PpaC | Manganese-dependent inorganic pyrophosphatase | Yes | C158 cons. | (K)SPTC158(+Cys)TEQDIAAAK(E) | 727.3190 | n.d. | 1452.6235 | 2 |
| A7Z657 | SerA | d-3-phosphoglycerate dehydrogenase (serine biosynthesis) | No | C410 cons. | (K)ISSNESGYDNC410(+BSH)ISVK(V) | 671.2758 | 626.8900 | 2010.8054 | 3 |
| A7Z7R9 | AroA | Phospho-2-dehydro-3-deoxyheptonate aldolase/chorismatemutase (chorismate biosynthesis) | No | C126 cons. | (R)FIVGPC126(+BSH)AVESYEQVAEVAAAAK(K) | 883.4087 | n.d. | 2647.2042 | 3 |
| A7Z612 | AroE | 3-phosphoshikimate 1-carboxyvinyltransferase (chorismate biosynthesis) | No | C79 not cons. | (K)GIDALC79(+BSH)EPDSLLDVGNSGTTIR(L) | 881.4009 | 836.7600 | 2641.1808 | 3 |
| (K)GIDALC79(+Cys)EPDSLLDVGNSGTTIR(L) | 789.0375 | n.d. | 2364.0906 | 3 | |||||
| A7Z0D1 | GuaB | Inosine-5′-monophosphate dehydrogenase (GMP biosynthesis) | Yes | C308 active site (cons.) forms thioimidate intermediate | (K)VGIGPGSIC308(+BSH)TTR(V) | 778.8491 | 712.0500 | 1555.6837 | 2 |
| A7Z8C1 | YumC | Ferredoxin—NADP reductase 2 (FAD-dependent Pyridine nucleotide-disulfide oxidoreductase) | Yes | C85 probably active site (cons.) | (K)FDQTIC85(+Cys)LEQAVESVEK(Q) | 653.3023 | n.d. | 1956.8851 | 3 |
| (K)FDQTIC85(+BSH)LEQAVESVEK(Q) | 745.6595 | 701.2400 | 2233.9568 | 3 | |||||
| A7Z7N3 | ThiI | Probable tRNA sulfur transferase (YtbJ homolog) | No | C81 not cons. | (K)C81(+BSH)ESKLEDIK(K) | 730.8164 | n.d. | 1459.6183 | 2 |
| (Thiamine biosynthesis) | |||||||||
| A7Z4X4 | CotE | Spore coat protein E | No | C113 cons. | (K)VLQQPNC113(+Cys)LEVTISPNGNK(I) | 691.6771 | n.d. | 2072.0094 | 3 |
| F4EAC4 | SufA | Chaperone involved in Fe-S cluster assembly | No | C102,104,120 cons: Fe-S-cluster | (K)NAGTPEEC120(+BSH)(-) | 608.7031 | 541.5800 | 1215.3916 | 2 |
| A7Z1M1 | Alr | Alanine racemase 1 (cell wall biosynthesis) | Yes | C63 not cons. | (K)AALEAGASC63(+BSH)LAVAILDEAISLR(K) | 851.7523 | 807.5500 | 2552.2351 | 3 |
| B. pumilus | |||||||||
| B4AEV7 | MetE | 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (methionine synthase) | No | C647,730 Zn-binding active site (cons.) | (R)FWVNPDC730(+BSH)GLK(T) | 787.8287 | 721.0000 | 1573.6428 | 2 |
| A8FIT1 | YwaA | Branched-chain amino-acid aminotransferase (valine, leucine, isoleucine biosynthesis) | No | C104 S-SCys by diamide in B. sub. | (R)LC104(+BSH)IPQIDTETVLEGLNELIR(I) | 889.1032 | 844.8300 | 2664.2878 | 3 |
| A8FGB2 | AroA | Phospho-2-dehydro-3-deoxyheptonate aldolase/chorismatemutase (chorismate biosynthesis) | No | C126 cons. | (R)FIVGPC126(+BSH)AVESYEQVAEVAAAAK(Q) | 883.4035 | n.d. | 2647.1887 | 3 |
| B4AN33 | Tuf | Elongation factor Tu | Yes | C83 in GTP-binding site 82–86 (cons.) | (R)HYAHVDC83(+BSH)PGHADYVK(N) | 527.7175 | 494.4800 | 2106.8408 | 4 |
| B4AF61 | MetI | Cystathionine-gamma synthase (CGS) (O-succinylhomoserine(thiol)-lyase) (homocysteine biosynthesis) | No | C335 cons. | (R)IANGVC369(+BSH)NK(L) | 607.7542 | 540.7300 | 1213.4939 | 2 |
| B4ANL1 | LuxS | S-ribosylhomocysteine lyase (homocysteine biosynthesis) | No | C84 essential for catalysis (cons.) C126 Fe-binding site (cons.) | (R)FC41(+BSH)QPNK(Q) | 566.7175 | 499.7200 | 1131.4204 | 2 |
| B4AF80 | ThiG | Thiazole synthase (Thiamine biosynthesis) | No | C92 cons. | (K)VEVIGC102(+BSH)SR(S) | 629.7681 | 562.9200 | 1257.5217 | 2 |
| B4AFT4 | KatX2 | Catalase | ? | No Cys cons. | (K)LLAIC461(+BSH)NFYR(A) | 503.5634 | 459.1000 | 1507.6684 | 3 |
| A8FCN5 | YkuU | 2-Cys peroxiredoxin | ? | C52, 169 cons.; C52 active site; C52–C169 catalytic disulfide | (R)VLQALQTGGLC169(+BSH)PANWKPGQK(T) | 835.7413 | 791.4100 | 2504.2022 | 3 |
| A8FHX4 | FliW | Flagellar assembly factor | No | C144 not cons. | (K)HLLEVASSC144(+BSH) | 677.7782 | 610.9300 | 1353.5418 | 2 |
| A8FG49 | CitZ | Citrate (Si)-synthase II (TCA cycle) | No | C195 cons. | (R)VC195(+BSH)VATLSDIYSGVTAAIGALK(G) | 816.7327 | 772.5300 | 2447.1762 | 3 |
| B. megaterium | |||||||||
| D5DIR6 | MetE | 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (methionine synthase) | No | C649,732 Zn-binding active site | (R)ALQVLDPALFWINPDC732(+BSH)GLK(T) | 837.0709 | 792.9500 | 2508.1908 | 3 |
| D5DER3 | PpaC | Manganese-dependent inorganic pyrophosphatase | Yes | C158 cons. | (K)SPTC158(+BSH)TDQDVAAAK(E) | 568.2314 | 523.7300 | 1701.6723 | 3 |
| (K)SPTC158(+Cys)TDQDVAAAK(E) | 713.3030 | n.d. | 1424.5915 | 2 | |||||
| D5D924 | GuaB | Inosine-5′-monophosphate dehydrogenase (GMP biosynthesis) | Yes | C308 active site, forms thioimidate intermediate (cons.) | (K)VGIGPGSIC308(+BSH)TTR(V) | 778.8499 | 712.1000 | 1555.6853 | 2 |
| S. carnosus | |||||||||
| B9DN54 | AroA | Phospho-2-dehydro-3-deoxyheptonate aldolase/chorismate mutase (chorismate biosynthesis) | No | C83, 125 cons; C83 catalytic site; C125 Fe-binding site | (K)SFIFGPC125(+BSH)SVESQEQVDK(V) | 765.9924 | 721.8800 | 2294.9553 | 3 |
| B9DKV8 | Tuf | Elongation factor Tu | No | C82 in GTP-binding site 81–85 | (R)HYAHVDC82(+BSH)PGHADYVK(N) | 527.7177 | 494.5300 | 2106.8417 | 4 |
| B9DM03 | GuaB | Inosine-5′-monophosphate dehydrogenase, (GMP biosynthesis) | No | C307 active site, forms thioimidate intermediate (cons.) | (K)VGIGPGSIC307(+BSH)TTR(I) | 519.5693 | 475.0700 | 1555.6862 | 3 |
| B9DNY0 | YphP | UPF0403 protein (thioredoxin-like protein) | No | C54 active site (CxC-motiv) cons. | (K)NVGKDETTFVVINSTC54(+BSH)GC56(+IAM)AAGLAR(P) | 720.5766 | 687.4300 | 2878.2773 | 4 |
| B9DJN8 | YtxJ | DUF2847 protein (thioredoxin-like protein) | No | C30 active site (TCPI-motiv) cons. | (K)HSNTC30(+BSH)PISANAYDQFNK(F) | 769.3155 | 724.9500 | 2304.9246 | 3 |
| B9DNK0 | DnaK | Chaperone protein | No | No Cys cons. | (K)IIGIDLGTTNSC15(+BSH)VAVLEGDEPK(V) | 880.7465 | 836.5500 | 2639.2176 | 3 |
| B9DNK1 | GrpE | Chaperone protein | No | No Cys cons. | (K)TYQAQC95(+BSH)VLTDILPTIDNIER(A) | 901.4220 | 856.9600 | 2701.2442 | 3 |
| B9DN51 | Ldh | l-lactate dehydrogenase | No | C72 cons. | (K)AGSYEDC72(+BSH)SDADLVVITAGAPQKPGETR(L) | 787.3511 | 754.4000 | 3145.3684 | 4 |
| Q9RGS6 | ThiM | Hydroxyethylthiazole kinase (thiamine biosynthesis) | No | C258 not cons. | (R)IDSDAVAENC258(+BSH)NLEEVK(-) | 715.6331 | 671.2800 | 2143.8775 | 3 |
| B9DMD5 | Sca_1625 | Putative aldehyde dehydrogenase family protein (similar to GbsA betaine aldehyde dehydrogenase) | ? | C279 active site (cons.) | (K)VYNNTGQVC279(+BSH)TAGTR(T) | 627.2645 | 582.8600 | 1878.7716 | 3 |
| (K)VYNNTGQVC279(+Cys)TAGTR(T) | 534.9047 | n.d. | 1601.6922 | 3 | |||||
| B9DN59 | YtpR | Similar to Phe-tRNA synthetase (YtpR homolog) | No | C126,167 cons. | (K)VNVGNEELQIVC126(+BSH)GAPNVEAGQK(V) | 888.7393 | 844.1200 | 2663.1961 | 3 |
| (K)VNVGNEELQIVC126(+Cys)GAPNVEAGQK(V) | 796.3812 | n.d. | 2386.1218 | 3 | |||||
| B9DNG1 | Dtd | d-tyrosyl-tRNA(Tyr) deacylase (YrvI homolog) | No | No Cys cons. | (R)LYEAFNEALC113(+Cys)QYGVEVK(T) | 698.9884 | n.d. | 2093.9434 | 3 |
| B9DLW2 | SceB | SceB precursor (SsaA homolog) | ? | C166 cons | (R)TSSGANYYTAGQC166(+BSH)TYYAFDR(A) | 879.0096 | 834.6200 | 2634.0069 | 3 |
| (R)TSSGANYYTAGQC166(+Cys)TYYAFDR(A) | 786.6523 | n.d. | 2356.9352 | 3 | |||||
| B9DN38 | Sca_1381 | Putative transaldolase | ? | C200 cons. | (R)ELLNVIQADEIGADIITC200(+BSH)PSGVISK(I) | 998.8192 | 954.2100 | 2993.4358 | 3 |
| B9DQ05 | TypA | Putative GTP-binding protein family protein (YlaG homolog) | No | C408 not cons. | (R)VQC408(+BSH)EVPQENAGAVIESLGQR(K) | 841.7123 | n.d. | 2522.1150 | 3 |
| B9DML0 | Sca_1554 | Putative ABC transporter ATP-binding protein | ? | No Cys cons. | (R)QIENC583(+IAM)EAEIEAC590(+BSH)EQK(I) | 730.6216 | 686.2800 | 2188.8431 | 3 |
| B9DM56 | Adk | Adenylate kinase | Yes | C130, 133, 150 cons., Zn-finger motif | (K)VDGVC150(+BSH)DLDGGK(L) | 491.8620 | 447.3800 | 1472.5642 | 3 |
Cytoplasmic protein extracts of B. subtilis, B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus were prepared in an urea-IAM-buffer, tryptic in-gel-digested, and analyzed in a LTQ Orbitrap-Velos™ mass spectrometer as described in the Materials and Methods section. Peptides with S-bacillithiolations were identified by the additional mass of 396 Da at Cys residues and the diagnostic neutral malate loss fragment ions that appeared as abundant ions in the fragment ion MS/MS spectra. Peptides with S-cysteinylation were identified with a mass difference of 119 Da at Cys residues. The table includes the Uniprot accession numbers and protein functions as derived from the UniprotKB database (http://uniprot.org/) and information about conserved Cys residues and Cys functions as revealed by the Conserved Domain Database CDD (34) (http://ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The table also includes the m/z of the precursor ions, m/z of the precursor malate loss ions (−134 Da), and the neutral molecular mass of the BSH-modified peptide and peptide charges. The Xcorr, ΔCn scores, and mass deviations of the S-bacillithiolated peptides and complete collision-induced diffraction (CID) MS/MS spectrum of all peptides and the b and y fragment ion series are given in Supplementary Figures S8 and S9 as follows: OhrR (S8A), MetE (S8B), YxjG (S8C), PpaC (S8D), SerA (S8E), AroA (S8H), Tuf (S8I), GuaB (S8J), YphP (S8F), YtxJ (S8G), YumC (S8K), ThrS (S8L), QueF (S8M), YwaA (S8N), MetI (S8O), LuxS (S8P), ThiG (S8U), KatX2 (S8S), YkuU (S8T), FliW (S8Q), CitZ (S8R), AroE (S8V), CotE (S8W), SufA (S8X), Alr (S8Y), ThiI (S8Z), DnaK (S9A), GrpE (S9B), Ldh (S9C), ThiM (S9D), Sca_1625 (S9E), YtpR (S9F), Dtd (S9G), SceB (S9H), Sca_1381 (S9I), TypA (S9J), Sca_1554 (S9K), and Adk (S9L). * indicates that these proteins with SSB sites have been identified in the previous study in B. subtilis (7) and are listed here for comparison. Bold texts indicate conserved S-bacillithiolated proteins that are identified in more than two of the four Bacillus species and S. carnosus in this study. #The YphP protein was identified in untreated and NaOCl-treated B. subtilis cells. ? indicates that it is unknown if this protein is essential. n.d. indicates that the malate-loss precursor ion was not determined.
IAM, iodoacetamide; LTQ, linear-trap quadrupole; MW, molecular weight.