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Identification of lipopeptide isoforms by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC

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Abstract

A three-stage linear gradient strategy using reverse-phase high-performance liquid chromatography (HPLC) was optimized for rapid, high-quality, and simultaneous purification of the lipopeptide isoforms of iturin, fengycin, and surfactin, which may differ in composition by only a single amino acid and/or the fatty acid residue. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was applied to detect the lipopeptides harvested from each reversed-phase HPLC peak. Amino acid analysis based on phenyl isothiocyanate derivatization was further used for confirmation of the amino acid species and molar ratio in a certain HPLC fraction. By this MALDI-TOF-MS/MS coupled with amino acid analysis, it was revealed that iturin at m/z 1,043 consists of a circular Asn-Tyr-Asn-Gln-Pro-Asn-Ser peptide and C14 β-OH fatty acid. Surfactin homologs from Bacillus subtilis THY-7 at m/z 1,030, 1,044, 1,058, and 1,072 possess a circular Glu-Leu-Leu-Val-Asp-Leu-Leu peptide and the β-OH fatty acid with a different length (C13–C16). Fengycin species at m/z 1,463 and 1,477 are homologs possessing the circular peptide Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile linked to a C16 or C17 γ-OH fatty acid, whereas fengycin at m/z 1,505 contains a Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile sequence with a Val instead of Ala at position 6. The method developed in this work provided an efficient approach for characterization of diverse lipopeptide isoforms from the iturin, fengycin, and surfactin families.

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References

  1. Liu XY, Yang SZ, Mu BZ (2009) Production and characterization of a C-15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilis HSO121. Process Biochem 44(10):1144–1151. doi:10.1016/j.procbio.2009.06.014

    Article  CAS  Google Scholar 

  2. Arima K, Kakinuma A, Tamura G (1968) Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun 31(3):488–494. doi:10.1016/0006-291x(68)90503-2

    Article  CAS  Google Scholar 

  3. Chen H, Wang L, Su CX, Gong GH, Wang P, Yu ZL (2008) Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis. Lett Appl Microbiol 47(3):180–186. doi:10.1111/j.1472-765X.2008.02412.x

    Article  CAS  Google Scholar 

  4. Wakayama S, Ishikawa F, Oishi K (1984) Mycocerein, a novel antifungal peptide antibiotic produced by Bacillus cereus. Antimicrob Agents Chemother 26(6):939–940

    Article  CAS  Google Scholar 

  5. Lee SC, Kim SH, Park IH, Chung SY, Choi YL (2007) Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity. Arch Microbiol 188(4):307–312. doi:10.1007/s00203-007-0250-9

    Article  CAS  Google Scholar 

  6. Desai JD, Banat IM (1997) Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev 61(1):47–64

    CAS  Google Scholar 

  7. Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53(5):495–508

    Article  CAS  Google Scholar 

  8. Das P, Mukherjee S, Sen R (2008) Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans. J Appl Microbiol 104(6):1675–1684. doi:10.1111/j.1365-2672.2007.03701.x

    Article  CAS  Google Scholar 

  9. Hiradate S, Yoshida S, Sugie H, Yada H, Fujii Y (2002) Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2. Phytochemistry 61(6):693–698. doi:10.1016/s0031-9422(02)00365-5

    Article  CAS  Google Scholar 

  10. Yu GY, Sinclair JB, Hartman GL, Bertagnolli BL (2002) Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biol Biochem 34(7):955–963. doi:10.1016/s0038-0717(02)00027-5

    Article  CAS  Google Scholar 

  11. Vanittanakom N, Loeffler W, Koch U, Jung G (1986) Fengycin - a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot 39(7):888–901

    Article  CAS  Google Scholar 

  12. Villegas-Escobar V, Ceballos I, Mira JJ, Argel LE, Peralta SO, Romero-Tabarez M (2013) Fengycin C Produced by Bacillus subtilis EA-CB0015. J Nat Prod 76(4):503–509. doi:10.1021/np300574v

    Article  CAS  Google Scholar 

  13. Bonmatin JM, Laprevote O, Peypoux F (2003) Diversity among microbial cyclic lipopeptides: Iturins and surfactins. Activity-structure relationships to design new bioactive agents. Comb Chem High Throughput Screen 6(6):541–556

    Article  CAS  Google Scholar 

  14. Hathout Y, Ho YP, Ryzhov V, Demirev P, Fenselau C (2000) Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis. J Nat Prod 63(11):1492–1496. doi:10.1021/np000169q

    Article  CAS  Google Scholar 

  15. Peypoux F, Pommier MT, Marion D, Ptak M, Das BC, Michel G (1986) Revised structure of mycosubtilin, a peptidolipid antibiotic from Bacillus subtilis. J Antibiot 39(5):636–641

    Article  CAS  Google Scholar 

  16. Volpon L, Besson F, Lancelin JM (1999) NMR structure of active and inactive forms of the sterol-dependent antifungal antibiotic bacillomycin L. Eur J Biochem 264(1):200–210. doi:10.1046/j.1432-1327.1999.00605.x

    Article  CAS  Google Scholar 

  17. Li YM, Yang SZ, Mu BZ (2010) The surfactin and lichenysin isoforms produced by Bacillus licheniformis HSN 221. Anal Lett 43(6):929–940. doi:10.1080/00032710903491047

    Article  CAS  Google Scholar 

  18. Bonmatin JM, Labbe H, Grangemard I, Peypoux F, Magetdana R, Ptak M, Michel G (1995) Production, isolation and characterization of [Leu4]- and [Ile4]surfactins from Bacillus subtilis. Lett Pept Sci 2(1):41–47. doi:10.1007/bf00122922

    Article  CAS  Google Scholar 

  19. Volpon L, Tsan P, Majer Z, Vass E, Hollosi M, Noguera V, Lancelin JM, Besson F (2007) NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics. Spectrochim Acta Part A 67(5):1374–1381. doi:10.1016/j.saa.2006.10.027

    Article  Google Scholar 

  20. Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS (2002) Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68(12):6210–6219. doi:10.1128/aem. 68.12.6210-6219.2002

    Article  CAS  Google Scholar 

  21. Sun LJ, Lu ZX, Bie XM, Lu FX, Yang SY (2006) Isolation and characterization of a co-producer of fengycins and surfactins, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi. World J Microbiol Biotechnol 22(12):1259–1266. doi:10.1007/s11274-006-9170-0

    Article  CAS  Google Scholar 

  22. Finking R, Marahiel MA (2004) Biosynthesis of nonribosomal peptides. Annu Rev Microbiol 58:453–488. doi:10.1146/annurev.micro.58.030603.123615

    Article  CAS  Google Scholar 

  23. Marahiel MA (2009) Working outside the protein-synthesis rules: insights into non-ribosomal peptide synthesis. J Pept Sci 15(12):799–807. doi:10.1002/psc.1183

    Article  CAS  Google Scholar 

  24. Chen CY, Baker SC, Darton RC (2006) Batch production of biosurfactant with foam fractionation. J Chem Technol Biotechnol 81(12):1923–1931. doi:10.1002/jctb.1625

    Article  CAS  Google Scholar 

  25. Mata-Sandoval JC, Karns J, Torrents A (1999) High-performance liquid chromatography method for the characterization of rhamnolipid mixtures produced by Pseudomonas aeruginosa UG2 on corn oil. J Chromatogr A 864(2):211–220. doi:10.1016/s0021-9673(99)00979-6

    Article  CAS  Google Scholar 

  26. Haddad NIA, Wang J, Mu BZ (2008) Isolation and characterization of a biosurfactant producing strain, Brevibacilis brevis HOB1. J Ind Microbiol Biotechnol 35(12):1597–1604. doi:10.1007/s10295-008-0403-0

    Article  CAS  Google Scholar 

  27. Yuan J, Raza W, Huang QW, Shen QR (2011) Quantification of the antifungal lipopeptide iturin A by high performance liquid chromatography coupled with aqueous two-phase extraction. Jo Chromatogr B 879(26):2746–2750. doi:10.1016/j.jchromb.2011.07.041

    Article  CAS  Google Scholar 

  28. Bidlingmeyer BA, Cohen SA, Tarvin TL (1984) Rapid analysis of amino acids using pre-column derivatization. J Chromatogr 336(1):93–104. doi:10.1016/s0378-4347(00)85133-6

    Article  CAS  Google Scholar 

  29. Cohen SA, Bidlingmeyer BA, Tarvin TL (1986) PITC derivatives in amino acid analysis. Nature 320(6064):769–770. doi:10.1038/320769a0

    Article  CAS  Google Scholar 

  30. Campanella L, Crescentini G, Avino P (1999) Simultaneous determination of cysteine, cystine and 18 other amino acids in various matrices by high-performance liquid chromatography. J Chromatogr A 833(2):137–145. doi:10.1016/s0021-9673(98)01023-1

    Article  CAS  Google Scholar 

  31. Harvey DJ (2003) Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates and glycoconjugates. Int J Mass Spectrom 226(1):1–35. doi:10.1016/s1387-3806(02)00968-5

    Article  CAS  Google Scholar 

  32. Sivapathasekaran C, Mukherjee S, Samanta R, Sen R (2009) High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium. Anal Bioanal Chem 395(3):845–854. doi:10.1007/s00216-009-3023-2

    Article  CAS  Google Scholar 

  33. Yanagi M, Yamasato K (1993) Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiol Lett 107(1):115–120

    Article  CAS  Google Scholar 

  34. Yang J, Sun L, Bai X, Zhou H (2002) Simultaneous determination of 18 amino acids by reversed-phase high performance liquid chromatography with precolumn phenylisothiocyanate derivatization. Se Pu 20(4):369–371

    CAS  Google Scholar 

  35. Liu X-Y, Yang S-Z, Mu B-Z (2008) Isolation and characterization of a C(12)-lipopeptide produced by Bacillus subtilis HSO 121. J Pept Sci 14(7):864–875. doi:10.1002/psc.1017

    Article  CAS  Google Scholar 

  36. Zabet-Moghaddam M, Heinzle E, Tholey A (2004) Qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices. Rapid Commun Mass Spectrom 18(2):141–148. doi:10.1002/rcm.1293

    Article  CAS  Google Scholar 

  37. Harrison AG (2003) Fragmentation reactions of protonated peptides containing glutamine or glutamic acid. J Mass Spectrom 38(2):174–187. doi:10.1002/jms.427

    Article  CAS  Google Scholar 

  38. Paizs B, Suhai S (2005) Fragmentation pathways of protonated peptides. Mass Spectrom Rev 24(4):508–548. doi:10.1002/mas.20024

    Article  CAS  Google Scholar 

  39. Wysocki VH, Tsaprailis G, Smith LL, Breci LA (2000) Special feature: commentary - mobile and localized protons: a framework for understanding peptide dissociation. J Mass Spectrom 35(12):1399–1406. doi:10.1002/1096-9888(200012)35:12<1399::aid-jms86>3.0.co;2-r

    Article  CAS  Google Scholar 

  40. Madonna AJ, Voorhees KJ, Taranenko NI, Laiko VV, Doroshenko VM (2003) Detection of cyclic lipopeptide biomarkers from Bacillus species using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 75(7):1628–1637. doi:10.1021/ac020506v

    Article  CAS  Google Scholar 

  41. Sen R (2008) Biotechnology in petroleum recovery: the microbial EOR. Prog Energy Combust 34(6):714–724. doi:10.1016/j.pecs.2008.05.001

    Article  CAS  Google Scholar 

  42. Bachmann RT, Johnson AC, Edyvean RGJ (2014) Biotechnology in the petroleum industry: an overview. Int Biodeterior Biodegrad 86:225–237. doi:10.1016/j.ibiod.2013.09.011

    Article  CAS  Google Scholar 

  43. Sachdev DP, Cameotra SS (2013) Biosurfactants in agriculture. Appl Microbiol Biotechnol 97(3):1005–1016. doi:10.1007/s00253-012-4641-8

    Article  CAS  Google Scholar 

  44. Mandal SM, Barbosa A, Franco OL (2013) Lipopeptides in microbial infection control: scope and reality for industry. Biotechnol Adv 31(2):338–345. doi:10.1016/j.biotechadv.2013.01.004

    Article  CAS  Google Scholar 

  45. Bockmuhl D (2012) Biosurfactants as antimicrobial ingredients for cleaning products and cosmetics. Tenside Surfactants Deterg 49(3):196–198

    Article  Google Scholar 

  46. Whang LM, Liu PWG, Ma CC, Cheng SS (2009) Application of rhamnolipid and surfactin for enhanced diesel biodegradation-effects of pH and ammonium addition. J Hazard Mater 164(2–3):1045–1050. doi:10.1016/j.jhazmat.2008.09.006

    Article  CAS  Google Scholar 

  47. Ongena M, Jacques P, Toure Y, Destain J, Jabrane A, Thonart P (2005) Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis. Appl Microbiol Biotechnol 69(1):29–38. doi:10.1007/s00253-005-1940-3

    Article  CAS  Google Scholar 

  48. Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16(3):115–125. doi:10.1016/j.tim.2007.12.009

    Article  CAS  Google Scholar 

  49. Kracht M, Rokos H, Ozel M, Kowall M, Pauli G, Vater J (1999) Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives. J Antibiot 52(7):613–619

    Article  CAS  Google Scholar 

  50. Dufour S, Deleu M, Nott K, Wathelet B, Thonart P, Paquot M (2005) Hemolytic activity of new linear surfactin analogs in relation to their physico-chemical properties. Biochimi Biophys Acta 1726(1):87–95. doi:10.1016/j.bbagen.2005.06.015

    Article  CAS  Google Scholar 

  51. Yang SZ, Wei DZ, Mu BZ (2006) Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism. J Biochem Biophys Methods 68(1):69–74. doi:10.1016/j.jbbm.2006.03.008

    Article  CAS  Google Scholar 

  52. Kwanyuen P, Burton JW (2010) A modified amino acid analysis using PITC derivatization for soybeans with accurate determination of cysteine and half-cystine. J Am Oil Chem Soc 87(2):127–132. doi:10.1007/s11746-009-1484-2

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the 973 National Key Basic Research Project (2013CB733600), the National Natural Science Foundation (no. 21176143), and the Tsinghua University Initiative Scientific Research Program (no. 20111081120).

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Correspondence to Huimin Yu.

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Yang, H., Li, X., Li, X. et al. Identification of lipopeptide isoforms by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC. Anal Bioanal Chem 407, 2529–2542 (2015). https://doi.org/10.1007/s00216-015-8486-8

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