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Identification of lipopeptides in Bacillus megaterium by two-step ultrafiltration and LC–ESI–MS/MS

Identification of lipopeptides in Bacillus megaterium by two-step ultrafiltration and LC–ESI–MS/MS A two-step ultrafiltration and liquid chromatography-electronic spray ionization-tandem mass spectrometry (LC–ESI– MS/MS) were applied to investigate the nature of lipopeptides in the methanol extract harvested from marine Bacillus megaterium. The structure of lipopeptide homologs were characterized by using collision-induced dissociation mass spectrometry analysis. Collectively, two kinds of linear fengycin A and B with a double bond were characterized for the first time besides the popular cyclic fengycin A and B with the difference in the amino acid at position 6 of the peptide moiety. It is worth noting that two kinds of linear isoforms of surfactins and two kinds of cyclic isoforms of esperin with a smaller peptide ring formed by the fifth l -Asp and β-hydroxy fatty acid were also separated and identi- fied in addition to the all seven kinds of cyclic isoforms of surfactins. Only one analogue (bacillomycin D) of iturin fam- ily was identified in this research. To our best knowledge, this is the first report that more than 40 variants of lipopep - tides from one strain of Bacillus were identified using our potent purification and identification methods. Keywords: Lipopeptide, Ultrafiltration, Bacillus megaterium, LC–ESI–MS, LC–ESI–MS/MS, Identification β-hydroxy fatty acid with a length of 14–18 carbons that Introduction can be saturated or unsaturated (Ongena and Jacques Lipopeptides produced by members of Bacillus as sec- 2008). Different strains of bacilli produce diverse types ondary metabolites are amphiphilic molecules with of cyclic lipopeptides. Related studies have shown that, hydrophilic and hydrophobic moieties (Ongena and some of the strains can produce only one family of lipo- Jacques 2008; Georgiou et  al. 1992). Surfactins, iturins peptides while some strains can produce two or all three and fengycins are the three most well-known fami- families of lipopeptides (Romero et  al. 2007; Yang et  al. lies of lipopeptides (Fig.  1). Surfactin is composed of an 2015; Pecci et  al. 2010). There is an increasing interest amphipathic, cyclic heptapeptide head group which is in lipopeptides because of their relatively nontoxic, bio- interlinked with a hydrophobic β-hydroxy fatty acid tail, degradable and unique structures which are suitable for comprising 12–16 carbon atoms (Hoefler et  al. 2012; their potential applications to many aspects of industry, Rosenberg et  al. 2016).The iturin family, represented by ranging from biotechnology to environmental cleanup iturin A, C, D, E, bacillomycin D, F, L, Lc and mycosub- (Jacques 2011). The complexity and high cost of purifica - tilin, are heptapeptides cyclized by a β-amino fatty acid tion is the most important limitation for the commercial chain containing 14–17 carbons (Romero et  al. 2007; use of lipopeptides. Yang et  al. 2015). Members of the fengycin family, rep- Ultrafiltration is highly suitable for the purification of resented by fengycin A and B are decapeptides with an lipopeptides from a series of extremely complex extract internal lactone ring in the peptidic moiety and with a of bacteria and impurity. If the material concentration is higher than critical micelle concentration (CMC), the lipopeptide monomers will gather to form micelles *Correspondence: kongqing@ouc.edu.cn School of Food Science and Engineering, Ocean University of China, with 2–3 times of molecular mass than monomers. But Yushan Road 5, 62 Building, Qingdao 266003, Shandong, China if methanol was added to micellar solution, micelles Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Ma et al. AMB Expr (2016) 6:79 Page 2 of 15 fengycins are linear lipopeptides which were seldom been reported. To our best knowledge, this is the first report 1 2 3 4 5 6 7 Rn-CH-CH2-CO-Glu -X1 -Leu -X2 -Asp -Leu -X3 that more than 40 variants of lipopeptides were identi- fied in a single microorganism. The purification method and identification results can provide basis for large-scale X =Ile,Leu or Val preparation and widely application of lipopeptides. X2=Ile,Leu or Val Materials and methods X =Ile,Leu or Val Bacterial strain and culture conditions The marine B. megaterium strain CGMCC7086 used 1 2 3 4 5 6 7 R -CH-CH -CO-Asn -Tyr -Asn -Gln -Pro -Asn -Ser n 2 in this work was previously isolated from the intes- tines of marine fish. For production of the lipopeptides, an inoculum of marine B. megaterium into 50  mL of NH Luria–Bertani medium in 250-mL Erlenmeyer flask and incubated at 37  °C for 16  h with shaking at 180  rpm. 1 2 3 4 5 6 R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Ala Five percent of the 16-h-old inoculum was seeded into a n 2 500-mL flask containing 200  mL fermentation medium 10 9 8 7 (1  L: 20  g glucose, 1  g yeast extract, 7  g NH NO , 7  g OH Ile -Tyr -Gln -Pro 4 3 NaCl, 0.78  g MgSO ·7H O, 0.5  g KCl, 1.5  g KH PO , 4 2 2 4 0.0004  g CuSO ·7H O, 0.005  g MnSO ·H O, 0.0002  g 4 2 4 2 FeSO ·7H O, pH 7.0), and the culture was incubated at 1 2 3 4 5 6 4 2 Rn-CH-CH2-CO-Glu -Orn -Tyr -Thr -Glu -Val the same condition for 72 h. 10 9 8 7 OH Ile -Tyr -Gln -Pro Extraction and purification of lipopeptides Fig. 1 Basic structures of representative members and diversity One thousand milliliters of the 72-h-old fermentation within the three lipopeptide families. a Basic structures of surfactin. broth of B. megaterium was centrifuged at 9000×g at 4 °C b Basic structures of iturin. c Basic structures of fengycin A. d Basic for 20 min (Hitachi, Tokyo, Japan). The supernatant fluid structures of fengycin B was collected and its pH was acidified to 2.0 with 6  M HCl and then incubated overnight at 4 °C for precipitat- ing lipopeptides. will recover into monomers. This feature can be used The precipitates were harvested by centrifuging again to process a two-step ultrafiltration for the purifica - at 10,000×g at 4 °C for 20 min. And then, the precipitates tion of lipopeptides by choosing appropriate aperture of were extracted twice with anhydrous methanol for 5  h ultrafiltration membrane (Isa et  al. 2007). Electrospray and the insoluble impurities were removed by centrifu- ionisation (ESI) tandem mass spectrometry was used to gation at 8000×g at 4  °C for 20  min. The methanol was identify lipopeptides homologs produced by B. licheni- evaporated using a vacuum rotary evaporator at 50  °C formis (Pecci et  al. 2010) and B. subtilis (Iatsenko et  al. to concentrate the lipopeptides and the resulting sample 2014). Surfactins, iturins and fengycins are all in the m/z was subsequently dissolved in a certain volume of Milli- range 1000–2000 that can be detected by ESI–MS (Yang Q water. et al. 2015; Sivapathasekaran et al. 2009). The lipopeptides were preliminarily purified through A strain of marine B. megaterium CGMCC7086 was two-step ultrafiltration by a Millipore tubular polysulfone previously isolated by our group from the intestine of ultrafiltration separation unit with membranes cut-off marine fish (Kong et al. 2010). In this work, lipopeptides molecules smaller than 30,000  Da. First step, ultrafiltra - were isolated and preliminarily purified from marine B. tion membrane separation unit was used to deal with megaterium through a two-step ultrafiltration. A liquid lipopeptides aqueous solutions and deep indentation chromatography-electronic spray ionization-tandem fluid was collected. Then, the pH of the mixture was mass spectrometry (LC–ESI–MS/MS) method was also adjusted to 7.0 using 1.0  M NaOH and added a certain developed to characterize the structure and composition volume of methanol with slow stirring for 2  h. Second of lipopeptide families. Collision-induced dissociation step, the same method was used and the upper filtration (CID) mass spectrometry analysis was used to character- liquid was collected. The upper filtration liquid was fur - ize the structure of lipopeptide homologs. Furthermore, ther concentrated using vacuum evaporator and subse- over 40 lipopeptides homologs including bacillomycin quently using vacuum freeze drying to obtain powdery D, surfactins, esperins and fengycins were identified. production of lipopeptides for further purification. The Of which, two broad types including surfactins and Ma et al. AMB Expr (2016) 6:79 Page 3 of 15 lipopeptides mixture was dissolved in Milli-Q water and filtered through a 0.2 μm membrane filter (PALL Gelman 5.87 Laboratory, New York, USA) to obtain a 500 μg/ml stock 6.39 solution for mass spectra analysis. 80 6.42 5.26 5.53 6.50 Liquid chromatography electrospray ionization mass 5.12 4.81 spectrometry (LC–ESI–MS) analysis of lipopeptides 8.11 40 8.69 8.05 The separation of the lipopeptide factions and all mass 8.17 6.94 7.17 8.63 9.22 spectrometry analyses were performed on a surveyor 9.12 8.40 20 4.68 9.38 8.46 HPLC on line with a LCQ Fleet Ion Trap mass spec- 9.63 trometer (Thermo Fisher Scientific, Waltham, MA, 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 USA) equipped with an electrospray ion source in posi- Time tive ion mode. LC–ESI–MS spectra were measured by passing the analysis through a Zorbax SB-C column 9.03 (Agilent, Santa Clara, USA) (2.1 × 100 mm, 3.5 μm par- 8.68 ticle size). 10.02 6.36 Lipopeptides were eluted by a component solvent sys- 9.51 10.84 tem that solvent A was acetonitrile and solvent B was 11.80 10.28 7.47 11.25 water, both injected with 0.1  % formic acid for facilitat- 60 ing protonation of the basic nitrogen compounds yielding 12.36 protonated molecules [M  +  H] . Two different elution programs were used with a flow rate of 200  μL/min and the column temperature was stayed at 35  °C. A 100  μL 6 7 8 9 10 11 12 aliquot was injected into system and gradient strategy for Time (min) surfactin was as follows: 0–3.5  min, 60  % A to 93  % A; Fig. 2 LC–ESI/MS analysis of the crude methanolic extract from the 3.5–20 min, 93 % A and 7 % B. The gradient strategy for marine B. megaterium strain. a Chromatograms of the surfactin and its both iturin and fengycin were as follows: 0–9  min, 45  % analogues, positive ion mode, TIC. b Chromatograms of the iturin and fengycin family, positive ion mode, TIC A to 55  % A; 9–20  min, 55  % A and 45  % B. The elec - trospray source was operated at a spray voltage of 4  kV, a capillary voltage of 35 V, and a capillary temperature of and 1050, as well as molecules [M  +  Na] m/z 1072, 325 °C; simultaneously, LC–MS full scan positive modes 1034, 1072, 1048, 1048, 1062, 1076 (1016), 1016, 1016, were performed from in the range from m/z 200 to 2000. 1030 (1030, 1030, 1030), 1090, 1044, 1044, 1044, 1030 ESI–MS/MS coupled with CID and the collision gas of (1030), 1058, 1030, 1058, 1058 (1044), 1044, 1072 (1044) helium were used for further identification of the amino and 1072 in positive modality. These data were shown in acid sequence. The selected precursor ions were acquired Fig. 3a–c. in an auto LC–ESI–MS/MS modalities and then the data The chromatogram for iturin and fengycin family were analyzed by Xcalibur 2.1 (Thermo Fisher Scientific (Fig. 2b) showed the main peaks from 6 to 13 min. In the Inc, Waltham, MA, USA). range 6–13  min, the main peaks of a series of lipopep- tides were eluted at retention time 6.36, 7.47, 8.68, 9.03, Results 9.51, 10.02, 10.28, 10.84, 11.25, 11.80 and 12.36  min, LC–ESI–MS analysis of lipopeptide extract obeying to protonated molecules [M  +  H] m/z 1031, To elute all putative lipopeptides, we used the designed 1045, 1467, 1481 (1059), 1495, 1509 (1073), 1451, 1465, elution program for surfactins and iturins as well as 1479 (1479) 1493 (1493) and 1495 (1495), and also cor- fengycins. The full scan LC–ESI–MS chromatogram for responding to sodiated molecules [M  +  Na] m/z 1053, surfactins (Fig.  2a) showed the main peaks from 4 to 1067, 1489, 1503 (1081), 1517, 1531 (1095), 1473, 1487, 11  min. In the range 4–11  min, the main peaks of sur- 1501 (1501) and 1515 (1505). It is noteworthy that the factin and its analogues were eluted at retention time retention time at 8.68, 9.03, 9.51, 10.02, 10.28, 10.84, (tR) 4.68, 4.81, 5.12, 5.26, 5.53, 5.87, 6.39, 6.42, 6.50, 6.94, 11.25 and 11.80  min, they were revealed corresponding 7.17, 8.05, 8.11, 8.17, 8.40, 8.46, 8.63, 8.69, 9.12, 9.22, 9.38 2+ to protonated molecules [M +  2H] m/z 734, 741, 748, and 9.63 min, consistenting to molecules [M +  H] m/z 755, 726, 733, 740 and 747. These results were shown in 1050, 1012, 1050, 1026, 1026, 1040, 1054 (994), 994, 994, Fig.  3d–f. Some other [M  +  H] were 1523, 1537 and 1008 (1008, 1008, 1008), 1068, 1022, 1022, 1022, 1008 2+ [M + 2H] were 717 and 762. (1008), 1036, 1008, 1036, 1036 (1022), 1022, 1050 (1022) Relative Abundance Relative Abundance Ma et al. AMB Expr (2016) 6:79 Page 4 of 15 1054.40 1040.44 a b 20000 1022.59 1026.34 18000 40000 1036.69 1068.29 1054.40 20000 1076.66 6000 1008.51 1012.30 10000 1090.69 994.49 0 0 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 500 600 700 800 900 1000 1100 1200 1300 1400 m/z m/z 1036.60 c 1031.48 1045.49 1059.43 1050.57 1044.55 1058.71 1022.49 1072.71 1073.65 1081.04 1008.48 500 950 1000 1050 1100 1150 850 900 950 1000 1050 1100 1150 m/z m/z 1481.65 e 1493.72 755.75 1495.65 1479.53 13000 1100 1509.68 1467.66 11000 748.87 747.55 734.54 741.93 1465.69 500 740.52 1523.67 733.68 1451.67 762.44 1537.67 3000 2000 726.51 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 m/z 600 650 700 750 800 850 900 950 1000 m/z Fig. 3 LC-ESI–MS lipopeptides spectrum. a–c Clusters of surfactin family. d Clusters of iturin family. e Clusters of fengycin family with single-charge molecule. f Clusters of fengycin family with double-charge molecular LC–ESI–MS/MS characterization of surfactin lipopeptides CID fragments, the sequence could be deduced as C 1 2 3 4 Figure  4a displayed the LC–ESI–MS/MS spectrum of β-hydroxyl fatty acid chain-Glu -Leu/Ile -Leu -Val - 5 6 7 the precursor ion at m/z 1022 with appearance of frag- Asp -Leu -Leu/Ile . Additionally, the LC–ESI–MS/MS ment ions at retention time 8.05  min, series of b and spectra of protonated ions found at m/z 994 at reten- y ions could be easily assigned, which meaned the ini- tion time 6.50  min (Additional file  1: Figure S1a), 1008 tial cleavage of protonated ester bond. The series of b at retention time 6.98 min (Additional file  1: Figure S1b), fragment ions at m/z 1022(−H O, 1004)  →  909  →  796  1036 at retention time 9.12  min (Additional file  1: Fig- →  681  →  582  →  469  →  356 were consistented with ure S1c) and 1050 at retention time 9.38 min (Additional 7 6 5 4 3 2 the losses of Leu/Ile -Leu -Asp -Val -Leu -Leu/Ile file  1: Figure S1d) with multiples of 14 Da (−CH ) differ - from the C terminus. Furthermore, the second typical ence, were proved to be homologs possessing the same set of y fragment ions contained the peptidic moiety amino acids sequence but different C , C , C and 12 13 15 inside the C-terminal product ions at m/z 1022(−H O, C β-OH fatty acids, respectively. These data were in 2 16 1004)  →  667(+H O, 685)  →  554  →  441 revealed the accordance with mass spectra of Sigma surfactin stand- 1 2 losses of C β-hydroxyl fatty acid chain-Glu -Leu/Ile - ard (S3523, Sigma-Aldrich, St. Louis, MO, USA) (data Leu from the precursor ion. According to these typical not shown). Intensity Intensity Intensity Intensity Intensity Intensity Ma et al. AMB Expr (2016) 6:79 Page 5 of 15 a b 685.33 685.38 100 100 90 90 80 80 70 70 60 60 582.20 50 50 582.28 1004.42 40 40 1004.52 554.00 891.28 666.96 30 469.01 30 356.12 796.41 554.42 20 20 451.02 441.04 909.52 536.12 778.38 695.56 356.09 536.02 681.28 923.22 10 10 810.23 0 0 400 500 600 700 800 900 1000 400 500 600 700 800 900 1000 m/z m/z 671.38 c d 685.28 681.53 990.52 1004.61 909.36 40 909.40 540.09 427.24 568.39 20 451.19 796.51 582.10 356.28 469.17 681.23 10 699.74 438.56 778.41 338.17 400 500 600 700 800 900 1000 m/z 400 500 600 700 800 900 1000 m/z 671.32 540.17 50 483.06 653.22 1004.21 20 695.23 352.02 905.03 300 400 500 600 700 800 900 1000 m/z Fig. 4 LC-ESI–MS/MS spectrum of the surfactin precursors. a–c Surfactin precursors ion [M + H] at m/z 1022 at retention time 8.05, 8.17, 8.11 min, respectively, containing a C14 β-hydroxy fatty acid chain. d Surfactin precursors ion [M + H] at m/z 1008 at retention time 8.40 min containing a Glu1-Leu/Ile2-Leu3-Val4-Asp5-Leu6-Val7 peptide and a C14 β-hydroxy fatty acid chain. e Surfactin precursors ion [M + H] at m/z 1022 at retention time 9.35 min containing a C15 β-hydroxy fatty acid chain The second peptide sequence was slightly differ- MS spectrum of species observed as protonated ion ent from the typical surfactins (Fig.  4b; Additional at m/z 1022 at retention time 8.17  min. In the same file  1: Figure S2). Figure  4b showed the LC–ESI–MS/ way, it was suggested that the precursor ion possessed Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 6 of 15 1 2 3 4 1 2 3 4 5 6 a peptide sequence of Glu -Leu/Ile -Leu -Leu/Ile - β-OH fatty acid-Glu -Val -Leu -Leu/Ile -Asp -Leu - 5 6 7 7 Asp -Leu -Val and a C β-hydroxyl fatty acid chain. Val , respectively (Fig. 5c, d). In addition, the LC–ESI–MS/MS spectra of proto- A seventh surfactin isoform of LC–ESI–MS/MS spec- nated ions found at m/z 994 at retention time 6.38 min trum of species observed as protonated ion was showed 1 2 3 (Additional file  1: Figure S2a), 1008 at retention time in Fig. 5e with a peptide sequence of Glu -Leu/Ile -Leu - 4 5 6 7 6.94 min (Additional file  1: Figure S2b), 1036 at reten- Leu/Ile -Asp -Leu -Leu/Ile and a C β-hydroxyl fatty tion time 8.63  min (Additional file  1: Figure S2c) and acid chain with no Val. 1050 at retention time 9.63 min (Additional file 1: Fig- Figure  6 displayed the LC–ESI–MS/MS spectrum of ure S2d) with multiples of 14  Da (−CH2) difference, the precursor ion at m/z 1050 at retention time 4.68 min. were proved to be homologs with the same amino acid It could be concluded that it was a kind of esperin with 2 3 4 sequence but different C , C , C and C β-OH fatty the sequence could be surmised as Val -Leu - Leu/Ile - 12 13 15 16 5 6 7 acids. Asp -Leu -Leu/Ile with a C β-OH fatty acid based on Figure  4c and Additional file  1: Figure S3 showed a its chromatographic behavior and fragmentation pattern. third surfactin isoforms of a series of homologs. Fig- In the same way, we can conclude that the sequence of ure  4c showed the LC–ESI–MS/MS spectrum of spe- the precursor ion at m/z 1050 at retention time 5.12 min 1 2 3 4 cies observed as protonated ion at m/z 1022 at retention was C β-OH fatty acid-Glu -Leu/Ile -Leu -Leu/Ile - 5 6 7 time 8.11  min. Which was suggested the precursor ion Asp -Leu -Val (Additional file  1: Figure S4), an esperin, 1 2 3 possessed a peptide sequence of Glu -Val -Leu -Leu/ too. 4 5 6 7 Ile -Asp -Leu -Leu/Ile and a C β-hydroxyl fatty acid As it is more, Fig.  7a showed the LC–ESI–MS/MS chain. Simultaneously, the LC–ESI–MS/MS spectra spectrum of species observed as protonated ion at m/z of protonated ions found at m/z 994 at retention time 1026 at retention time 5.26 min. We can conclude it was 6.42  min (Additional file  1: Figure S3a), 1008 at reten- a linear surfactin with a peptide sequence of Glu -Leu/ 2 3 4 5 6 7 tion time 6.89  min (Additional file  1: Figure S3b), 1036 Ile -Leu -Val -Asp -Leu -Leu/Ile and a C β-hydroxyl at retention time 8.69  min (Additional file  1: Figure fatty acid chain. In addition, the protonated ion m/z at S3c) and with multiples of 14  Da (−CH ) difference, 1012 at retention time 4.79 min (Additional file  1: Figure were proved to be homologs with the same amino acid S5a), 1040 at retention time 5.89  min (Additional file  1: sequence but different C , C and C β-OH fatty Figure S5b), 1054 at retention time 6.36 min (Additional 12 13 15 acids. file  1: Figure S5c) and 1068 at retention time 7.19  min The fourth surfactin isoforms of LC–ESI–MS/MS (Additional file  1: Figure S5d) were assigned as homologs spectrum of species observed as protonated ion were with C , C , C , C β-OH fatty acid. Using the same 11 13 14 15 showed in Fig.  4d, e. Figure  4d showed fragment ions method, we can easily identify the protonated ion at m/z of precursor ion m/z 1008 at retention time 8.40  min. 1026 at the retention time 5.53  min was a linear surfac- 1 2 3 Then the sequence could be concluded based on the tin possessing a peptide sequence of Glu -Leu/Ile -Leu - 1 4 5 6 7 fragmentation profile as C β-OH fatty acid—Glu - Val -Asp -Leu -Val and a C β-hydroxyl fatty acid chain 14 14 2 3 4 5 6 7 Leu/Ile -Leu -Val -Asp -Leu -Val . In addition to this, (Fig. 7b). a protonated ion m/z at 1022 at retention time 9.35 min The protonated ion species of surfactin family con - was assigned as homologs with C β-OH fatty acid taining with surfactins, esperins and linear surfactins (Fig. 4e). isoforms characterized from the culture supernatants of Figure  5b showed the LC–ESI–MS/MS spectrum of marine B. megatherium are summarized in Table 1. species observed as protonated ion at m/z 1022 at reten- tion time 8.11 min. Then the sequence could be surmised LC–ESI–MS/MS characterisation of iturin lipopeptides 1 2 3 4 5 6 as C β-OH fatty acid-Glu -Val -Leu -Val -Asp -Leu - Figure  8 displayed the LC–ESI–MS/MS spectrum of the Leu/Ile . Thus, a protonated ion m/z at 1008 at retention precursor ion at m/z 1031 with appearance of fragment time 8.46 min was assigned as homologs with C β-OH ions at retention time 6.98  min. A series y fragment at 7 6 5 fatty acid (Fig. 5a). m/z 317 (+H O, 335)(Thr -Ser -Glu ),528 (−H O, 510) 2 2 7 6 5 4 3 7 6 In the same way, we inferred the sequence of the pre- (Thr -Ser -Glu -Pro -Asn ), 691 (−H O, 673)(Thr -Ser - 5 4 3 2 1 7 6 5 cursor ion at m/z 1008 at retention time 8.40  min and Glu -Pro -Asn -Tyr -Asn ) and 805 (Thr -Ser -Glu - 4 3 2 1 1022 at retention time 9.16  min were C β-OH fatty Pro -Asn -Tyr -Asn -β-amino fatty acid) were found in 1 2 3 4 5 6 7 + acid-Glu -Val -Leu -Leu/Ile -Asp -Leu -Val and C the MS spectrum of m/z 1031. The b fragment ion at 15 2 Ma et al. AMB Expr (2016) 6:79 Page 7 of 15 ab 671.30 671.21 90 90 990.66 80 80 60 60 649.30 877.96 1004.46 50 50 782.67 40 568.36 796.35 582.22 441.14 909.37 441.02 455.35 469.04 891.49 20 20 554.27 423.24 554.19 10 10 370.26 917.25 450 500 550 600 650 700 750 800 850 900 950 1000 400 500 600 700 800 900 1000 m/z m/z cd 671.29 671.38 100 681.53 441.15 990.52 653.61 554.26 40 1004.57 909.36 568.25 30 451.22 923.41 695.53 810.46 796.51 20 582.29 469.26 356.28 352.19 792.25 699.74 300 400 500 600 700 800 900 1000 400 500 600 700 800 900 1000 m/z m/z 990.51 568.19 50 681.16 536.36 30 423.38 782.61 328.06 554.13 895.34 441.24 20 667.24 342.13 455.20 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z Fig. 5 LC-ESI–MS/MS spectrum of the surfactin precursors. a, b Surfactin precursors ion [M + H] at m/z 1008 at retention time 8.46 and 9.22 min containing a Glu1-Val2-Leu3-Val4-Asp5-Leu6-Leu/Ile7 peptide and a C14 and C15 β-hydroxy fatty acid chain, respectively. c, d Surfactin precursors ion [M + H] at m/z 1008 at retention time 8.40 and 9.16 min containing a Glu1-Val2-Leu3-Leu/Ile4-Asp5-Leu6-Val7 peptide and a C14 and C15 β-hydroxy fatty acid chain, respectively. e Surfactin precursor ions ion [M + H] at m/z 1008 at retention time 6.89 min containing a Glu1-Leu/Ile2- Leu3-Leu/Ile4-Asp5-Leu6-Leu/Ile7 peptide and a C12 β-hydroxy fatty acid chain Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 8 of 15 The protonated ion species of bacillomycin D homologs 806.51 characterized from the culture supernatants of marine B. megaterium are summarized in Table 1. 708.53 LC/ESI–MS/MS characterisation of fengycin lipopeptides 937.55 Figure  9a displayed the LC–ESI–MS/MS spectrum of 30 the precursor ion at m/z 1479 with appearance of frag- 464.29 ment ions at retention time 12.63  min. The results 595.42 10 365.44 482.31 1032.71 919.36 + showed the appearance of b product ions at m/z 300 400 500 600 700 800 900 1000 1100 1200 1479  →  1177(−H O, 1159)  →  1080  →  952  →  676(− m/z 4 5 H O, 658) were assigned as the losses of (Thr -Glu - 6 7 8 9 10 + Ala )-Pro -Gln -(Tyr -Ile ) and the y product ions at m/z 1210 → 1081 → 967 were corresponded to the neu- 1 2 + tral losses of side-chain Glu –Orn . These fragment ions Fig. 6 LC-ESI–MS/MS spectrum of esperin precursors ion [M + H] at 3 4 were formed upon cleavage at Tyr and Thr in the mid- m/z 1050 at retention time 4.68 min containing a Glu1-Leu/Ile2-Leu3- Leu/Ile4-Asp5-Leu6-Val7 peptide and a C16 β-hydroxy fatty acid chain dle of molecule. A third series fragment ions at m/z 1408 6 5 6 and 1279 meant the losses of Ala and Glu - Ala , respec- tively. This fragment series suggested there was another 6 7 1 2 3 ring cleavage site between Ala and Pro of fengycin A m/z 617 (β-amino fatty acid-Asn -Tyr -Asn ) and 948 1 2 3 4 5 6 homolog. (β-amino fatty acid-Asn -Tyr -Asn -Pro -Glu -Ser ) were Figure  9b showed the LC–ESI–MS/MS spectrum of found, too. According to these typical CID fragments, 2+ the precursor ion [M  +  2H] at m/z 740 with appear- the sequence could be deduced as C β-amino fatty acid 1 2 3 4 5 6 7 ance of fragment ions at retention time 12.69  min. Dis- chain-Asn -Tyr -Asn -Pro -Glu -Ser -Thr , which was sociation yielded b ions at m/z 1479  →  1177  →  1080  assigned as bacillomycin D homologs. →  952  →  789  →  676 were surmised as the losses of The MS/MS spectrum of other protonated precursor 4 5 6 7 8 9 10 + (Thr -Glu -Ala )-Pro -Gln -Tyr -Ile and y ions at m/z ions at m/z 1045 at retention time 7.95  min (Additional 1081 and 967 were corresponded to the neutral loses of file  1: Figure S6) also yielded similar profile of product β-hydroxyl fatty acid chain-Glu and β-hydroxyl fatty ions was proved to be homologs possessing the same 1 2 acid chain-Glu -Orn , respectively. From the product amino acid sequence but different C β-amino fatty + 2+ ions, [M + H] at m/z 1479 and [M + 2H] at m/z 734 acids. ab x10 2.8 2.6 2 x10 568.5 2.4 1.1 671.6 2.2 356.3 455.2 0.9 685.3 1.8 0.8 667.2 1.6 0.7 1.4 0.6 469.4 342.0 1.2 0.5 582.1 0.4 441.2 0.3 0.8 451.2 227.0 328.1 0.2 0.6 681.5 778.0 0.1 0.4 554.3 227.4 0.2 199.3 -0.1 100 200 300 400 500 600 700 800 900 150 200 250 300 350 400 450 500 550 600 650 700 750 800 m/z m/z Fig. 7 LC-ESI–MS/MS spectrum of the linear surfactin precursors. a Linear surfactin precursors ion [M + H] at m/z 1026 at retention time 5.26 min containing a Glu1-Leu/Ile2-Leu3-Val4-Asp5-Leu6-Leu/Ile7 peptide and a C13 β-hydroxy fatty acid chain. b Linear surfactin precursor ions [M + H] at m/z 1026 at retention time 5.53 min containing a Glu1-Leu/Ile2-Leu3-Val4-Asp5-Leu6-Val7 peptide and a C14 β-hydroxy fatty acid chain Intensity Relative Abundance(%) Intensity Ma et al. AMB Expr (2016) 6:79 Page 9 of 15 Table 1 Assignment of the structures of lipopeptides by LC–ESI–MS/MS in this study Peak Mass (M/S) Rt (min) Family Assignment Sequence 1 2 3 4 5 6 7 1 994 6.50 Surfactin C [M + H] 12 R -CH-CH -CO-Glu -Leu/Ile -Leu -Val -Asp -Leu -Leu/Ile n 2 2 1008 6.98 C [M + H] 3 1022 8.05 C [M + H] 4 1036 9.12 C [M + H] 5 1050 9.38 C [M + H] 1 2 3 4 5 6 7 6 994 6.38 C [M + H] 12 R -CH-CH -CO-Glu -Leu/Ile -Leu -Leu/Ile -Asp -Leu -Val n 2 7 1008 6.94 C [M + H] 8 1022 8.17 C [M + H] 9 1036 8.63 C [M + H] 10 1050 9.63 C [M + H] 1 2 3 4 5 6 7 11 994 6.42 C [M + H] 12 R -CH-CH -CO-Glu -Val -Leu -Leu/Ile -Asp -Leu -Leu/Ile n 2 12 1008 6.89 C [M + H] 13 1022 8.11 C [M + H] 14 1036 8.69 C [M + H] 1 2 3 4 5 6 7 16 1008 8.40 C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Val -Asp -Leu -Val 14 n 2 17 1022 9.35 C [M + H] 1 2 3 4 5 6 7 18 1008 8.46 C [M + H] 14 R -CH-CH -CO-Glu -Val -Leu -Val -Asp -Leu -Leu/Ile n 2 19 1022 9.22 C [M + H] 1 2 3 4 5 6 7 20 1008 8.40 Surfactin C [M + H] R -CH-CH -CO-Glu -Val -Leu -Leu/Ile -Asp -Leu -Val 14 n 2 21 1022 9.16 C [M + H] 1 2 3 4 5 6 7 22 1008 6.89 C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Leu/Ile -Asp -Leu -Leu/Ile 12 n 2 1 2 3 4 5 6 7 23 1050 4.68 Esperin C [M + H] 16 R -CH-CH -CO-Glu -Val -Leu -Leu/Ile -Asp -Leu -Leu/Ile -COOH n 2 1 2 3 4 5 6 7 24 1050 5.12 C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Leu/Ile -Asp -Leu -Val -COOH 16 n 2 1 2 3 4 5 7 25 1012 4.81 Surfactin Linear C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Val -Asp -Leu6-Leu/Ile -COOH n 2 OH 26 1026 5.26 Linear C [M + H] 27 1040 5.87 Linear C [M + H] 28 1054 6.39 Linear C [M + H] 29 1068 7.17 Linear C [M + H] 1 2 3 4 5 6 7 30 1026 5.53 Surfactin Linear C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Val -Asp -Leu -Val -COOH 14 n 2 OH Ma et al. AMB Expr (2016) 6:79 Page 10 of 15 Table 1 continued Peak Mass (M/S) Rt (min) Family Assignment Sequence 1 2 3 4 5 6 7 31 1031 6.98 Bacillomycin D C [M + H] 14 R -CH-CH -CO-Asn -Tyr -Asn -pro -Glu -Ser -Thr n 2 NH 32 1045 7.95 C [M + H] 1 2 3 4 5 6 33 1451 10.94 Fengycin A C [M + H] 15 R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Ala n 2 10 9 8 7 OH Ile -Tyr -Gln -Pro 34 1465 10.82 C [M + H] 35 1479 12.63 C [M + H] 2+ 36 740 12.69 C [M + 2H] 37 1493 13.01 C [M + H] 2+ 38 747 15.09 C [M + 2H] 1 2 3 4 5 6 39 1479 12.71 Fengycin B C [M + H] 15 R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Val n 2 10 9 8 7 OH Ile -Tyr -Gln -Pro 2+ 40 740 12.61 C [M + 2H] 41 1493 12.92 C [M + H] 2+ 42 747 13.15 C [M + 2H] 1 2 3 4 5 6 7 8 9 10 43 1467 8.66 Fengycin A Linear C [M + H] R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Ala -Pro -Gln -Tyr -Ile -COOH 15 n 2 OH 44 1481 8.83 Linear C [M + H] 45 1495 12.36 Linear C [M + H] 1 2 3 4 5 6 7 8 9 10 46 1495 12.36 Fengycin B Linear C [M + H] 15 R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile -COOH n 2 OH 47 1509 13.87 Linear C [M + H] were with the same structure, it could be concluded that 1013.50 1 2 it was a kind of fengycin A with the sequence Glu –Orn - 3 4 5 6 7 8 9 10 805.09 Tyr -Thr -Glu -Ala -Pro -Gln -Tyr -Ile with a C β- 335.01 OH fatty acid based on its chromatographic behavior and 60 673.36 fragmentation pattern. 492.40 In addition, the protonated ion m/z at 1451 at reten- 617.16 510.82 30 599.26 tion time 10.94  min (Additional file  1: Figure S7a), 1465 948.55 at retention time 10.82  min (Additional file  1: Figure S7b), 1493 at retention time 13.01 min (Additional file  1: 400 500 600 700 800 900 1000 2+ Figure S7c) and [M + 2H] at m/z 747 at retention time m/z 15.09  min (Additional file  1: Figure S7d) with multiples of 14  Da difference in their molecular ion species were proved to be Fengycin A homologs with the same amino acid sequence but different C , C , C and C β-OH 15 16 18 18 fatty acids chain. Fig. 8 LC-ESI–MS/MS spectrum of the bacillomycin D precursors ion [M + H] at m/z 1031 at retention time 6.98 min containing a A second fengycin isoforms of LC–ESI–MS/MS spec- Asn1-Tyr2-Asn3-pro4-Glu5-Ser6-Thr7 peptide and a C14 β-amino fatty trum of species observed as protonated ion were showed acid chain on Fig.  9c, d and Additional file  1: Figure S8. Then the Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 11 of 15 ab 967.39 1080.95 952.38 40 1081.71 1408.24 30 676.53 789.49 952.44 1279.58 1159.21 658.40 20 1081.49 967.67 1177.58 1210.83 1080.50 1279.02 500 600 700 800 900 1000 1100 1200 1300 1400 400 500 600 700 800 900 1000 1100 1200 m/z m/z cd 995.48 1109.28 1109.65 991.57 814.37 775.43 1216.16 662.29 1145.60 832.39 731.33 502.56 20 389.13 20 226.44 1238.78 775.59 1163.62 1048.74 938.44 832.39 938.44 644.66 749.27 995.61 1145.62 385.25 10 499.32 10 895.06 503.21 500 600 700 800 900 1000 1100 1200 1300 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z m/z Fig. 9 LC-ESI–MS/MS spectrum of the fengycin precursors. a Fengycin A precursors ion [M + H] at m/z 1479 at retention time 12.63 min contain- ing a Glu1-Orn2-Tyr3-Thr4-Glu5-Ala6-Pro7-Gln8-Tyr9-Ile10 peptide and a C17 β-hydroxy fatty acid chain. b Fengycin A precursors ion [M + 2H]2 at m/z 740 at retention time 12.69 min containing a C17 β-hydroxy fatty acid chain. c Fengycin B precursors ion [M + H] at m/z 1493 at retention time 12.92 min containing a Glu1-Orn2-Tyr3-Thr4-Glu5-Val6-Pro7-Gln8-Tyr9-Ile10 peptide and a C16 β-hydroxy fatty acid chain. d Fengycin B precursors 2+ ion [M + 2H] at m/z 747 at retention time 13.15 min containing a C16 β-hydroxy fatty acid chain 2+ sequence of Fig.  9c could be concluded based on the Figure S8a) and [M + 2H] at m/z 740 at retention time 1 2 fragmentation profile as C β-OH fatty acid-Glu –Orn - 12.61 min (Additional file  1: Figure S8b) were assigned as 3 4 5 6 7 8 9 10 Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile . Figure  9d fengycin B homologs with C β-OH fatty acid. 2+ showed fragment ions of precursor ion [M  +  2H] at Figure  10a showed the LC–ESI–MS/MS spectrum of m/z 747 at retention time 13.15  min. It turned out that species observed as protonated ion at m/z 1495 at reten- it has the same structure with m/z 1493 at retention tion time 12.36 min. We concluded it is a linear fengycin 1 2 3 4 5 time 12.92 min. In addition to this, a protonated ion m/z A with a peptide sequence of Glu -Orn -Tyr -Thr -Glu - 6 7 8 9 10 at 1479 at retention time 12.71  min (Additional file  1: Ala -Pro -Gln -Tyr -Ile and a C β-hydroxyl fatty acid Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 12 of 15 a b 1013.22 493.19 40 40 922.17 521.21 1054.19 30 975.30 1363.63 1127.41 747.41 656.38 20 975.30 1200.57 20 1200.57 1363.63 775.24 1381.49 876.46 967.24 483.45 1054.19 592.38 721.45 10 1381.49 500 600 700 800 900 1000 1100 1200 1300 1400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z m/z Fig. 10 LC-ESI–MS/MS spectrum of the linear fengycin precursors. a Linear fengycin A precursors ion [M + H] at m/z 1495 containing a Glu1- Orn2-Tyr3-Thr4-Glu5-Ala6-Pro7-Gln8-Tyr9-Ile10 peptide and a C17 β-hydroxy fatty acid chain with a double bond. b Linear fengycin B precursors ion [M + H] at m/z 1495 containing a Glu1-Orn2-Tyr3-Thr4-Glu5-Val6-Pro7-Gln8-Tyr9-Ile10 peptide and a C15 β-hydroxy fatty acid chain with a double bond chain with a double bond based on its chromatographic families of molecules were successively separated and behavior and fragmentation pattern. The protonated ion characterised by LC–ESI–MS in positive full scan mode. m/z at 1467 at retention time 8.66 min (Additional file  1: Surfactin family contains more than 20 different lipo - Figure S9a) and 1481 at retention time 8.79  min (Addi- peptides such as lichenysin, pumilacidin, esperin and tional file  1: Figure S9b) with characteristic fragment ions surfactin. Esperin is coincided with those established sur- m/z 967 and 1081 were assigned as linear fengycin A factin for the composition and the amino acid residues, homologs with C and C β-OH fatty acid with a double except that there is a smaller peptide ring formed by the 15 16 bond, respectively. fifth l-Asp in the esperin, and not by the seventh Leu- The LC–ESI–MS/MS spectrum of species observed residue like surfactin (Kalinovskaya et  al. 1995; Thomas as protonated ion at m/z 1495 at retention time and Ito 1969). Surfactins have the common following 12.36  min had a second series of fragmentation ions structural traits: a heptapeptide with a chiral sequence were showed that it contained a peptide sequence of LLDLLDL interlinked with a β-hydroxy fatty acid and 1 2 3 4 5 6 7 8 9 10 Glu -Orn -Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile with a d-Leu in position 3 and 6 and a l-Asp in position and a C β-hydroxyl fatty acid chain with a double 5. Amino acid residues in position 2, 4 and 7 belong to bond (Fig.  10b). Besides, a protonated ion m/z at 1509 the amino acids group including Val, Leu and Ile (Jacques at retention time 13.87  min with characteristic frag- 2011; Hue et  al. 2001). With a Gln in the position 1 are ment ions m/z 995 was assigned as linear fengycin B named lichenysin which are different from surfactin with homologs with C β-OH fatty acid with a double bond the Glu in the position 1 (Qiu et  al. 2014). Some of the (Additional file 1 : Figure S10). isoforms had the same m/z by ESI–MS, but they were in The protonated ion species of fengycin family con - different HPLC fraction. Thus, it is necessary to precisely taining with fengycin A, B and linear fengycin isoforms identify their structure by MS/MS, especially for the characterized from the culture supernatants of marine B. amino acid sequence of the peptide portion of the mol- megaterium are summarized in Table 1. ecules (Yang et al. 2015). According to the results of LC–MS, ten major frac- Discussion tions with different molecular weight were used as pre - LC–ESI–MS was performed in order to investigate the cursor ions for further ESI–MS/MS analysis. In details, nature of the compounds in the methanol extract after the precursor ions at m/z 994, 1008, 1022, 1036 and 1050 a two-step ultrafiltration. Different families of lipopep - were hypothesized to be a series of homologue molecules tides with surfactins, fengycins and iturins can be eluted of surfactins with 14 or multiples of 14  Da difference in by 85–100, 50–70, 40–50  % acetonitrile in water, respec- their molecular ion species. tively, in reversed phase high performance liquid chroma- From the results of LC–ESI–MS/MS of Figs.  4 and 5, tography (RP-HPLC) system (Yang et al. 2015). The three the presence of fragment ions at m/z 685, 671 or 681 are Relative Abundance(%) Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 13 of 15 the common peaks in CID spectrum of surfactin isoforms (Maget-Dana and Peypoux 1994). Bacillomycin D, closely ion. The amino acids sequences at m/z 671, 685 and 681 related variants with iturin, is famous for the high activity 2 3 4 5 6 7 + are [(H)Leu/Ile -Leu -Val -Asp -Leu -Val (OH)  +  H] / against Aspergillus flavus (Moyne et al. 2001). According 2 3 4 5 6 7 + [(H)Val -Leu -Val -Asp -Leu -Leu/Ile (OH)  +  H] /[(H) to the results of LC–MS, the most intense fractions with 2 3 4 5 6 7 + Val -Leu -Leu/Ile -Asp -Leu -Val (OH) +  H] , [(H)Leu/ different molecules at m/z 1031 and 1045 were used as 2 3 4 5 6 7 + 2 Ile -Leu -Val -Asp -Leu - Leu/Ile (OH)  +  H] /[(H)Val - precursor ions by ESI–MS/MS analyzes for further pep- 3 4 5 6 7 + + Leu -Leu/Ile -Asp -Leu -Leu/Ile (OH)  +  H] /[(H)Leu/ tide sequence determination and some others [M +  H] 2 3 4 5 6 7 + Ile -Leu -Leu/Ile -Asp -Leu -Val (OH)  +  H] and [(H) at m/z 1059 and 1073. They were hypothesized to be a 2 3 4 5 6 7 + Leu/Ile -Leu -Leu/Ile -Asp -Leu -Leu/Ile (OH)  +  H] , series of homologous molecules of bacillomycin D with respectively. u Th s, the fragment ions at m/z 685, 671 or multiples of 14  Da difference in their molecular ion 681 are characteristic marker ions for identification of species. surfactins. Fengycin homologs are divided into two different types Through LC–ESI–MS, the chromatogram for surfactin (fengycin A and B) by their amino acid sequence. The family showed two unique peaks at retention time 4.68 fengycin A has the Ala at position 6, while Val for fengy- + 9 and 5.12 min corresponding to molecules [M +  H] m/z cin B (Vater et  al. 2002). Fengycins (plipastatin if Tyr is 1050. Their retention times were much less than the sur - D-configured) (Raaijmakers et  al. 2010), the configura - factin standard time, thus, according to the principle of tion of amino acid sequence is LDLDLDDLLDL with a similar miscibility, it showed that these molecules with lactone bond connecting l-Tyr to l Ile, includes fengy- stronger polarity. Figure 6 showed the dissociation yielded cins A, B and relevant homologs with the difference b ions at m/z 1050 (−H O, 1032) → 919 → 806 → 708  in the amino acid at position 6. They are macrolactone →  595  →  482  →  383 (−H O, 365) were surmised as rings with the Tyr side chain at position 3 of the peptide 7 6 5 4 the losses of (OH)Leu/Ile -Leu -Asp(-OH) -Leu/Ile - sequence and the C-terminal residue yielding an internal 3 2 Leu -Val from the C terminus. Therefore, we can infer ring by an ester bond. 1 2 3 4 5 its amino acids sequence is Glu -Val -Leu -Leu/Ile -Asp - From the above, the results (Fig. 9a, b; Additional file  1: 6 7 Leu -Leu/Ile , which is the same with surfactin. But, its Figure S7) showed the appearance of product ions at m/z fragment ions didn’t have the characteristic fragment ion 1081 and 967 from a series of precursor ions and the m/z 685 (Figs.  4, 5) which can be inferred it wasn’t sur- results (Fig. 9c, d and Additional file  1: Figure S8) showed factin. Simultaneously, the peak from m/z 806 to 708 in the appearance of product ions at m/z 1109 and 995 from Fig.  6 was formed by loss of the Asp(−OH) from mol- a series of precursor ions. The molecular weights of two ecule, which confirmed that the carboxyl group of Asp group peaks (1081 and 1109, 967 and 995) were differed and the hydroxyl group of the aliphatic part formed the by 28 Da, suggesting a difference in the amino acid com - ester structure in the esperin molecule, and this is another position of the peptide (Ala or Val). Thus, m/z 1080 and evidence of our inferences. The most noteworthy is that 967 are characteristic ions for fengycin A and m/z 1109 the fragment ions of it at m/z 450 were matched with the and 995 are characteristic ions for fengycin B with the + 7 6 5 losses of H (OH)Val -Leu -Asp -C β-OH fatty acid information of specific octapeptide ring ions. from the C terminus which further improved the carboxyl The result (Fig.  10a) showed the b fragment ions at group of Asp and the hydroxyl group of the aliphatic part m/z 1495(−H O, 1477)  →  1363  →  1200  →  1072(-H O, 2 2 formed the ester structure in the esperin molecule. 1054) →  975 →  904(+H O, 922) →  775 →  674(−H O, 2 2 According to LC–ESI–MS, before the elution of sur- 656) → 511(−H O, 493) matched with the losses of Tyr - 4 5 6 7 8 9 10 + factins (4.5–7.2 min), several peaks appeared that corre- Thr -Glu -Ala -Pro -Gln -Tyr -Ile . Its y fragment ions spond to unknown compounds at m/z 1012 at retention at m/z 985(−H O, 967) → 721 → 592 → 521 meant the 3 4 5 6 time 4.79  min, 1026 at retention time 5.26  min, 1026 at losses of (Tyr -Thr )-Glu -Ala in the middle of precursor retention time 5.53 min, 1040 at retention time 5.89 min, ion and the peak of 967 is the characteristic fragment ion 1054 at retention time 6.36  min and 1068 at retention of fengycin A. It was 16 Da bigger than m/z 1479. We con- time 7.19  min. What a coincidence is that, compared cluded it was a linear fengycin A with a peptide sequence 1 2 3 4 5 6 7 8 9 10 with the surfactins at m/z 994, 1008, 1022, 1036 and of Glu -Orn -Tyr -Thr -Glu -Ala -Pro -Gln -Tyr -Ile 1050, respectively, the m/z precursor molecules are 18 Da and a C β-hydroxyl fatty acid chain with a double bond. bigger than surfactins correspondingly. As we know, the The result (Fig.  10b) showed the b fragment ions at m/z relative molecular mass of the H O was just 18. We can 1495(−H O, 1477)  →  1363  →  1200  →  1072(−H O, 2 2 2 conclude they were linear surfactins based on their chro- 1054) → 975 → 876 → 747 → 483 revealed the losses of 3 4 5 6 7 8 9 10 matographic behavior and fragmentation pattern. Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile from the C ter- Though iturin has the limited antiviral and antibac - minus. Its y fragment ions at m/z 1127 → 1013 meaned terial activity, but it displays strong antifungal activity the losses of Orn , suggesting the precursor ion (a linear Ma et al. AMB Expr (2016) 6:79 Page 14 of 15 1 2 Acknowledgements fengycin B) possessed a peptide sequence of Glu -Orn - 3 4 5 6 7 8 9 10 Dr. Yejun Han from Institute of Process Engineering, Chinese Academy of Sci- Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile and a C ences, is appreciated for helping improve this manuscript. β-hydroxyl fatty acid chain with a double bond. Competing interests LC–ESI–MS/MS was performed after a two-step The authors declare that they have no competing interests. ultrafiltration in order to investigate the nature of the compounds in the methanol extract of marine B. mega- Ethics approval and consent to participate This article does not contain any studies concerned with experimentation on terium. The three families of molecules were successively human or animals. separated and characterised by LC–ESI–MS in positive full scan mode. LC–ESI–MS/MS analysis precisely eluci- Funding This study was funded by the National Natural Science Foundation of China dated the different amino sequence of isoforms of iturin, (No. 31471657). surfactin and fengycin families. Collectively, we have established seven kinds of cyclic isoforms of surfactin Received: 7 September 2016 Accepted: 8 September 2016 with a series of homologs (Table 1). The typical difference among these surfactins is amino acid residues in posi- tion 2, 4 and 7 belong to the amino acids group including Val, Leu and Ile. Ile or Leu is difficult to distinguish with References mass spectrometry. It is worth noting that a variety of lin- Georgiou G, Lin SC, Sharma MM. Surface-active compounds from microorgan- ear isoforms of surfactin were identified with relatively isms. Nat Biotechnol. 1992;10(1):60–5. Hoefler BC, Gorzelnik KV, Yang JY, Hendricks N, Dorrestein PC, Straight PD. high abundance. In addition to this, two kinds of cyclic Enzymatic resistance to the lipopeptide surfactin as identified through isoforms of esperin were separated and identified, which imaging mass spectrometry of bacterial competition. Proc Natl Acad Sci was consistent with others (Kalinovskaya et  al. 1995). USA. 2012;109(32):13082–7. Hue N, Serani L, Laprévote O. Structural investigation of cyclic peptidolipids The peptide sequence of bacillomycin D homologs which from Bacillus subtilis by high-energy tandem mass spectrometry. Rapid exhibited potent inhibitory effect on A. flavus were also Commun Mass Spectrom. 2001;15(3):203–9. identified. In this study, in addition to the popular cyclic Iatsenko I, Yim JJ, Schroeder FC, Sommer RJ. Bacillus subtilis GS67 protects C. elegans from Gram-positive pathogens via fengycin-mediated microbial fengycin A and B with the difference in the amino acid antagonism. Curr Biol. 2014;24(22):2720–7. at position 6 of the peptide moiety, two kinds of linear Isa MHM, Coraglia DE, Frazier RA, Jauregi P. Recovery and purification of fengycin A and B with a double bond were characterized surfactin from fermentation broth by a two-step ultrafiltration process. J Membr Sci. 2007;296:51–7. for the first time. Jacques P. Surfactin and other lipopeptides from Bacillus spp. In: Soberón- In summary, this is the first report of an Bacillus strain Chávez G, editor. Biosurfactants. Berlin: Springer-Verlag; 2011. p. co-producing so many variants of lipopeptides including 57–91. Kalinovskaya NI, Kuznetsova TA, Rashkes YV, Mil’grom YM, Mil’grom EG, Willis cyclic surfactin, linear surfactin, esperin, bacillomycin RH, Wood AI, Kurtz HA, Carabedian C, Murphy P, Elyakov GB. Surfactin-like D, cyclic fengycin A, linear fengycin A, cyclic fengycin B structures of five cyclic depsipeptides from the marine isolate of Bacillus and linear fengycin B. The structure of different lipopep - pumilus. Russ Chem Bull. 1995;44(5):951–5. Kong Q, Shan SH, Liu QZ, Wang XD, Yu FT. Biocontrol of Aspergillus flavus on tides has a good reference for the future research. peanut kernels by use of a strain of marine Bacillus megaterium. Int J Food Microbiol. 2010;139(1–2):31–5. Additional file Maget-Dana R, Peypoux F. Iturins, a special class of poreforming lipo- peptides: biological and physicochemical properties. Toxicology. 1994;87(1–3):151–74. Additional file 1. Additional figures. Moyne AL, Shelby R, Cleveland TE, Tuzun S, Bacillomycin D. An iturin with antifungal activity against Aspergillus flavus. J Appl Microbiol. 2001;90(4):622–9. Abbreviations Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease LC–ESI–MS/MS: liquid chromatography-electronic spray ionization-tandem biocontrol. Trends Microbiol. 2008;16(3):115–25. mass spectrometry; CID: collision-induced dissociation; CMC: critical micelle Pecci Y, Rivardo F, Martinotti MG, Allegrone G. LC/ESI-MS/MS characterization concentration; CGMCC: China General Microbiological Culture Collection of lipopeptide biosurfactants produced by Bacillus licheniformis V9T14 Center; tR: retention time; RP-HPLC: reversed phase high performance liquid strain. J Mass Spectrom. 2010;45(7):772–8. chromatography. Qiu YM, Xiao F, Wei XT, Wen ZY, Chen SW. Improvement of lichenysin produc- tion in Bacillus licheniformis by replacement of native promoter of Authors’ contributions lichenysin biosynthesis operon and medium optimization. Appl Microbiol YXM, QK, YLC, CQ and YJC contributed to the conception and design of the Biotechnol. 2014;98(21):8895–903. experiments. YXM, QK, RHL and GHZ performed the experiments, analyzed the Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M. Natural functions of data and drafted the manuscript. All authors contributed in the final approval. lipopeptides from Bacillus and Pseudomonas: more than surfactants and All authors read and approved the final manuscript. antibiotics. FEMS Microbiol Rev. 2010;34:1037–62. Romero D, de Vicente A, Rakotoaly RH, Dufour SE, Veening JW, Arrebola Author details E, Cazorla FM, Kuipers OP, Paguot M, Pérez-García A. The iturin and School of Food Science and Engineering, Ocean University of China, Yushan fengycin families of lipopeptides are key factors in antagonism of Road 5, 62 Building, Qingdao 266003, Shandong, China. Department Bacillus subtilis towards Podosphaera fusca. Mol Plant Microbe Interact. of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann 2007;20(4):430–40. Arbor, MI, USA. Ma et al. AMB Expr (2016) 6:79 Page 15 of 15 Rosenberg G, Steinberg N, Oppenheimer-Shaanan Y, Olender T, Doron S, Ben- Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS. Matrix-assisted Ari J, Sirota-Madi A, Bloom-Ackermann Z, Kolodkin-Gal I. Not so simple, laser desorption ionization-time of flight mass spectrometry of lipo - not so subtle: the interspecies competition between Bacillus simplex and peptide biosurfactants in whole cells and culture filtrates of Bacillus Bacillus subtilis and its impact on the evolution of biofilms. npj Biofilms subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol. Microb. 2016;2:15027. 2002;68(12):6210–9. Sivapathasekaran C, Mukherjee S, Samanta R, Sen R. High performance liquid Yang H, Li X, Li X, Yu HM, Shen ZY. Identification of lipopeptide isoforms chromatography purification of biosurfactant isoforms produced by a by MALDY-TOF-MS/MS based on the simultaneous purification marine bacterium. Anal Bioanal Chem. 2009;395(3):845–54. of iturin, fengycin, and surfactin by RP-HPLC. Anal Bioanal Chem. Thomas DW, Ito T. The revised structure of the peptide antibiotic esperin, 2015;407(9):2529–42. established by mass spectrometry. Tetrahedron. 1969;25(9):1985–90. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AMB Express Springer Journals

Identification of lipopeptides in Bacillus megaterium by two-step ultrafiltration and LC–ESI–MS/MS

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Copyright © 2016 by The Author(s)
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
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Abstract

A two-step ultrafiltration and liquid chromatography-electronic spray ionization-tandem mass spectrometry (LC–ESI– MS/MS) were applied to investigate the nature of lipopeptides in the methanol extract harvested from marine Bacillus megaterium. The structure of lipopeptide homologs were characterized by using collision-induced dissociation mass spectrometry analysis. Collectively, two kinds of linear fengycin A and B with a double bond were characterized for the first time besides the popular cyclic fengycin A and B with the difference in the amino acid at position 6 of the peptide moiety. It is worth noting that two kinds of linear isoforms of surfactins and two kinds of cyclic isoforms of esperin with a smaller peptide ring formed by the fifth l -Asp and β-hydroxy fatty acid were also separated and identi- fied in addition to the all seven kinds of cyclic isoforms of surfactins. Only one analogue (bacillomycin D) of iturin fam- ily was identified in this research. To our best knowledge, this is the first report that more than 40 variants of lipopep - tides from one strain of Bacillus were identified using our potent purification and identification methods. Keywords: Lipopeptide, Ultrafiltration, Bacillus megaterium, LC–ESI–MS, LC–ESI–MS/MS, Identification β-hydroxy fatty acid with a length of 14–18 carbons that Introduction can be saturated or unsaturated (Ongena and Jacques Lipopeptides produced by members of Bacillus as sec- 2008). Different strains of bacilli produce diverse types ondary metabolites are amphiphilic molecules with of cyclic lipopeptides. Related studies have shown that, hydrophilic and hydrophobic moieties (Ongena and some of the strains can produce only one family of lipo- Jacques 2008; Georgiou et  al. 1992). Surfactins, iturins peptides while some strains can produce two or all three and fengycins are the three most well-known fami- families of lipopeptides (Romero et  al. 2007; Yang et  al. lies of lipopeptides (Fig.  1). Surfactin is composed of an 2015; Pecci et  al. 2010). There is an increasing interest amphipathic, cyclic heptapeptide head group which is in lipopeptides because of their relatively nontoxic, bio- interlinked with a hydrophobic β-hydroxy fatty acid tail, degradable and unique structures which are suitable for comprising 12–16 carbon atoms (Hoefler et  al. 2012; their potential applications to many aspects of industry, Rosenberg et  al. 2016).The iturin family, represented by ranging from biotechnology to environmental cleanup iturin A, C, D, E, bacillomycin D, F, L, Lc and mycosub- (Jacques 2011). The complexity and high cost of purifica - tilin, are heptapeptides cyclized by a β-amino fatty acid tion is the most important limitation for the commercial chain containing 14–17 carbons (Romero et  al. 2007; use of lipopeptides. Yang et  al. 2015). Members of the fengycin family, rep- Ultrafiltration is highly suitable for the purification of resented by fengycin A and B are decapeptides with an lipopeptides from a series of extremely complex extract internal lactone ring in the peptidic moiety and with a of bacteria and impurity. If the material concentration is higher than critical micelle concentration (CMC), the lipopeptide monomers will gather to form micelles *Correspondence: kongqing@ouc.edu.cn School of Food Science and Engineering, Ocean University of China, with 2–3 times of molecular mass than monomers. But Yushan Road 5, 62 Building, Qingdao 266003, Shandong, China if methanol was added to micellar solution, micelles Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Ma et al. AMB Expr (2016) 6:79 Page 2 of 15 fengycins are linear lipopeptides which were seldom been reported. To our best knowledge, this is the first report 1 2 3 4 5 6 7 Rn-CH-CH2-CO-Glu -X1 -Leu -X2 -Asp -Leu -X3 that more than 40 variants of lipopeptides were identi- fied in a single microorganism. The purification method and identification results can provide basis for large-scale X =Ile,Leu or Val preparation and widely application of lipopeptides. X2=Ile,Leu or Val Materials and methods X =Ile,Leu or Val Bacterial strain and culture conditions The marine B. megaterium strain CGMCC7086 used 1 2 3 4 5 6 7 R -CH-CH -CO-Asn -Tyr -Asn -Gln -Pro -Asn -Ser n 2 in this work was previously isolated from the intes- tines of marine fish. For production of the lipopeptides, an inoculum of marine B. megaterium into 50  mL of NH Luria–Bertani medium in 250-mL Erlenmeyer flask and incubated at 37  °C for 16  h with shaking at 180  rpm. 1 2 3 4 5 6 R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Ala Five percent of the 16-h-old inoculum was seeded into a n 2 500-mL flask containing 200  mL fermentation medium 10 9 8 7 (1  L: 20  g glucose, 1  g yeast extract, 7  g NH NO , 7  g OH Ile -Tyr -Gln -Pro 4 3 NaCl, 0.78  g MgSO ·7H O, 0.5  g KCl, 1.5  g KH PO , 4 2 2 4 0.0004  g CuSO ·7H O, 0.005  g MnSO ·H O, 0.0002  g 4 2 4 2 FeSO ·7H O, pH 7.0), and the culture was incubated at 1 2 3 4 5 6 4 2 Rn-CH-CH2-CO-Glu -Orn -Tyr -Thr -Glu -Val the same condition for 72 h. 10 9 8 7 OH Ile -Tyr -Gln -Pro Extraction and purification of lipopeptides Fig. 1 Basic structures of representative members and diversity One thousand milliliters of the 72-h-old fermentation within the three lipopeptide families. a Basic structures of surfactin. broth of B. megaterium was centrifuged at 9000×g at 4 °C b Basic structures of iturin. c Basic structures of fengycin A. d Basic for 20 min (Hitachi, Tokyo, Japan). The supernatant fluid structures of fengycin B was collected and its pH was acidified to 2.0 with 6  M HCl and then incubated overnight at 4 °C for precipitat- ing lipopeptides. will recover into monomers. This feature can be used The precipitates were harvested by centrifuging again to process a two-step ultrafiltration for the purifica - at 10,000×g at 4 °C for 20 min. And then, the precipitates tion of lipopeptides by choosing appropriate aperture of were extracted twice with anhydrous methanol for 5  h ultrafiltration membrane (Isa et  al. 2007). Electrospray and the insoluble impurities were removed by centrifu- ionisation (ESI) tandem mass spectrometry was used to gation at 8000×g at 4  °C for 20  min. The methanol was identify lipopeptides homologs produced by B. licheni- evaporated using a vacuum rotary evaporator at 50  °C formis (Pecci et  al. 2010) and B. subtilis (Iatsenko et  al. to concentrate the lipopeptides and the resulting sample 2014). Surfactins, iturins and fengycins are all in the m/z was subsequently dissolved in a certain volume of Milli- range 1000–2000 that can be detected by ESI–MS (Yang Q water. et al. 2015; Sivapathasekaran et al. 2009). The lipopeptides were preliminarily purified through A strain of marine B. megaterium CGMCC7086 was two-step ultrafiltration by a Millipore tubular polysulfone previously isolated by our group from the intestine of ultrafiltration separation unit with membranes cut-off marine fish (Kong et al. 2010). In this work, lipopeptides molecules smaller than 30,000  Da. First step, ultrafiltra - were isolated and preliminarily purified from marine B. tion membrane separation unit was used to deal with megaterium through a two-step ultrafiltration. A liquid lipopeptides aqueous solutions and deep indentation chromatography-electronic spray ionization-tandem fluid was collected. Then, the pH of the mixture was mass spectrometry (LC–ESI–MS/MS) method was also adjusted to 7.0 using 1.0  M NaOH and added a certain developed to characterize the structure and composition volume of methanol with slow stirring for 2  h. Second of lipopeptide families. Collision-induced dissociation step, the same method was used and the upper filtration (CID) mass spectrometry analysis was used to character- liquid was collected. The upper filtration liquid was fur - ize the structure of lipopeptide homologs. Furthermore, ther concentrated using vacuum evaporator and subse- over 40 lipopeptides homologs including bacillomycin quently using vacuum freeze drying to obtain powdery D, surfactins, esperins and fengycins were identified. production of lipopeptides for further purification. The Of which, two broad types including surfactins and Ma et al. AMB Expr (2016) 6:79 Page 3 of 15 lipopeptides mixture was dissolved in Milli-Q water and filtered through a 0.2 μm membrane filter (PALL Gelman 5.87 Laboratory, New York, USA) to obtain a 500 μg/ml stock 6.39 solution for mass spectra analysis. 80 6.42 5.26 5.53 6.50 Liquid chromatography electrospray ionization mass 5.12 4.81 spectrometry (LC–ESI–MS) analysis of lipopeptides 8.11 40 8.69 8.05 The separation of the lipopeptide factions and all mass 8.17 6.94 7.17 8.63 9.22 spectrometry analyses were performed on a surveyor 9.12 8.40 20 4.68 9.38 8.46 HPLC on line with a LCQ Fleet Ion Trap mass spec- 9.63 trometer (Thermo Fisher Scientific, Waltham, MA, 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 USA) equipped with an electrospray ion source in posi- Time tive ion mode. LC–ESI–MS spectra were measured by passing the analysis through a Zorbax SB-C column 9.03 (Agilent, Santa Clara, USA) (2.1 × 100 mm, 3.5 μm par- 8.68 ticle size). 10.02 6.36 Lipopeptides were eluted by a component solvent sys- 9.51 10.84 tem that solvent A was acetonitrile and solvent B was 11.80 10.28 7.47 11.25 water, both injected with 0.1  % formic acid for facilitat- 60 ing protonation of the basic nitrogen compounds yielding 12.36 protonated molecules [M  +  H] . Two different elution programs were used with a flow rate of 200  μL/min and the column temperature was stayed at 35  °C. A 100  μL 6 7 8 9 10 11 12 aliquot was injected into system and gradient strategy for Time (min) surfactin was as follows: 0–3.5  min, 60  % A to 93  % A; Fig. 2 LC–ESI/MS analysis of the crude methanolic extract from the 3.5–20 min, 93 % A and 7 % B. The gradient strategy for marine B. megaterium strain. a Chromatograms of the surfactin and its both iturin and fengycin were as follows: 0–9  min, 45  % analogues, positive ion mode, TIC. b Chromatograms of the iturin and fengycin family, positive ion mode, TIC A to 55  % A; 9–20  min, 55  % A and 45  % B. The elec - trospray source was operated at a spray voltage of 4  kV, a capillary voltage of 35 V, and a capillary temperature of and 1050, as well as molecules [M  +  Na] m/z 1072, 325 °C; simultaneously, LC–MS full scan positive modes 1034, 1072, 1048, 1048, 1062, 1076 (1016), 1016, 1016, were performed from in the range from m/z 200 to 2000. 1030 (1030, 1030, 1030), 1090, 1044, 1044, 1044, 1030 ESI–MS/MS coupled with CID and the collision gas of (1030), 1058, 1030, 1058, 1058 (1044), 1044, 1072 (1044) helium were used for further identification of the amino and 1072 in positive modality. These data were shown in acid sequence. The selected precursor ions were acquired Fig. 3a–c. in an auto LC–ESI–MS/MS modalities and then the data The chromatogram for iturin and fengycin family were analyzed by Xcalibur 2.1 (Thermo Fisher Scientific (Fig. 2b) showed the main peaks from 6 to 13 min. In the Inc, Waltham, MA, USA). range 6–13  min, the main peaks of a series of lipopep- tides were eluted at retention time 6.36, 7.47, 8.68, 9.03, Results 9.51, 10.02, 10.28, 10.84, 11.25, 11.80 and 12.36  min, LC–ESI–MS analysis of lipopeptide extract obeying to protonated molecules [M  +  H] m/z 1031, To elute all putative lipopeptides, we used the designed 1045, 1467, 1481 (1059), 1495, 1509 (1073), 1451, 1465, elution program for surfactins and iturins as well as 1479 (1479) 1493 (1493) and 1495 (1495), and also cor- fengycins. The full scan LC–ESI–MS chromatogram for responding to sodiated molecules [M  +  Na] m/z 1053, surfactins (Fig.  2a) showed the main peaks from 4 to 1067, 1489, 1503 (1081), 1517, 1531 (1095), 1473, 1487, 11  min. In the range 4–11  min, the main peaks of sur- 1501 (1501) and 1515 (1505). It is noteworthy that the factin and its analogues were eluted at retention time retention time at 8.68, 9.03, 9.51, 10.02, 10.28, 10.84, (tR) 4.68, 4.81, 5.12, 5.26, 5.53, 5.87, 6.39, 6.42, 6.50, 6.94, 11.25 and 11.80  min, they were revealed corresponding 7.17, 8.05, 8.11, 8.17, 8.40, 8.46, 8.63, 8.69, 9.12, 9.22, 9.38 2+ to protonated molecules [M +  2H] m/z 734, 741, 748, and 9.63 min, consistenting to molecules [M +  H] m/z 755, 726, 733, 740 and 747. These results were shown in 1050, 1012, 1050, 1026, 1026, 1040, 1054 (994), 994, 994, Fig.  3d–f. Some other [M  +  H] were 1523, 1537 and 1008 (1008, 1008, 1008), 1068, 1022, 1022, 1022, 1008 2+ [M + 2H] were 717 and 762. (1008), 1036, 1008, 1036, 1036 (1022), 1022, 1050 (1022) Relative Abundance Relative Abundance Ma et al. AMB Expr (2016) 6:79 Page 4 of 15 1054.40 1040.44 a b 20000 1022.59 1026.34 18000 40000 1036.69 1068.29 1054.40 20000 1076.66 6000 1008.51 1012.30 10000 1090.69 994.49 0 0 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 500 600 700 800 900 1000 1100 1200 1300 1400 m/z m/z 1036.60 c 1031.48 1045.49 1059.43 1050.57 1044.55 1058.71 1022.49 1072.71 1073.65 1081.04 1008.48 500 950 1000 1050 1100 1150 850 900 950 1000 1050 1100 1150 m/z m/z 1481.65 e 1493.72 755.75 1495.65 1479.53 13000 1100 1509.68 1467.66 11000 748.87 747.55 734.54 741.93 1465.69 500 740.52 1523.67 733.68 1451.67 762.44 1537.67 3000 2000 726.51 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 m/z 600 650 700 750 800 850 900 950 1000 m/z Fig. 3 LC-ESI–MS lipopeptides spectrum. a–c Clusters of surfactin family. d Clusters of iturin family. e Clusters of fengycin family with single-charge molecule. f Clusters of fengycin family with double-charge molecular LC–ESI–MS/MS characterization of surfactin lipopeptides CID fragments, the sequence could be deduced as C 1 2 3 4 Figure  4a displayed the LC–ESI–MS/MS spectrum of β-hydroxyl fatty acid chain-Glu -Leu/Ile -Leu -Val - 5 6 7 the precursor ion at m/z 1022 with appearance of frag- Asp -Leu -Leu/Ile . Additionally, the LC–ESI–MS/MS ment ions at retention time 8.05  min, series of b and spectra of protonated ions found at m/z 994 at reten- y ions could be easily assigned, which meaned the ini- tion time 6.50  min (Additional file  1: Figure S1a), 1008 tial cleavage of protonated ester bond. The series of b at retention time 6.98 min (Additional file  1: Figure S1b), fragment ions at m/z 1022(−H O, 1004)  →  909  →  796  1036 at retention time 9.12  min (Additional file  1: Fig- →  681  →  582  →  469  →  356 were consistented with ure S1c) and 1050 at retention time 9.38 min (Additional 7 6 5 4 3 2 the losses of Leu/Ile -Leu -Asp -Val -Leu -Leu/Ile file  1: Figure S1d) with multiples of 14 Da (−CH ) differ - from the C terminus. Furthermore, the second typical ence, were proved to be homologs possessing the same set of y fragment ions contained the peptidic moiety amino acids sequence but different C , C , C and 12 13 15 inside the C-terminal product ions at m/z 1022(−H O, C β-OH fatty acids, respectively. These data were in 2 16 1004)  →  667(+H O, 685)  →  554  →  441 revealed the accordance with mass spectra of Sigma surfactin stand- 1 2 losses of C β-hydroxyl fatty acid chain-Glu -Leu/Ile - ard (S3523, Sigma-Aldrich, St. Louis, MO, USA) (data Leu from the precursor ion. According to these typical not shown). Intensity Intensity Intensity Intensity Intensity Intensity Ma et al. AMB Expr (2016) 6:79 Page 5 of 15 a b 685.33 685.38 100 100 90 90 80 80 70 70 60 60 582.20 50 50 582.28 1004.42 40 40 1004.52 554.00 891.28 666.96 30 469.01 30 356.12 796.41 554.42 20 20 451.02 441.04 909.52 536.12 778.38 695.56 356.09 536.02 681.28 923.22 10 10 810.23 0 0 400 500 600 700 800 900 1000 400 500 600 700 800 900 1000 m/z m/z 671.38 c d 685.28 681.53 990.52 1004.61 909.36 40 909.40 540.09 427.24 568.39 20 451.19 796.51 582.10 356.28 469.17 681.23 10 699.74 438.56 778.41 338.17 400 500 600 700 800 900 1000 m/z 400 500 600 700 800 900 1000 m/z 671.32 540.17 50 483.06 653.22 1004.21 20 695.23 352.02 905.03 300 400 500 600 700 800 900 1000 m/z Fig. 4 LC-ESI–MS/MS spectrum of the surfactin precursors. a–c Surfactin precursors ion [M + H] at m/z 1022 at retention time 8.05, 8.17, 8.11 min, respectively, containing a C14 β-hydroxy fatty acid chain. d Surfactin precursors ion [M + H] at m/z 1008 at retention time 8.40 min containing a Glu1-Leu/Ile2-Leu3-Val4-Asp5-Leu6-Val7 peptide and a C14 β-hydroxy fatty acid chain. e Surfactin precursors ion [M + H] at m/z 1022 at retention time 9.35 min containing a C15 β-hydroxy fatty acid chain The second peptide sequence was slightly differ- MS spectrum of species observed as protonated ion ent from the typical surfactins (Fig.  4b; Additional at m/z 1022 at retention time 8.17  min. In the same file  1: Figure S2). Figure  4b showed the LC–ESI–MS/ way, it was suggested that the precursor ion possessed Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 6 of 15 1 2 3 4 1 2 3 4 5 6 a peptide sequence of Glu -Leu/Ile -Leu -Leu/Ile - β-OH fatty acid-Glu -Val -Leu -Leu/Ile -Asp -Leu - 5 6 7 7 Asp -Leu -Val and a C β-hydroxyl fatty acid chain. Val , respectively (Fig. 5c, d). In addition, the LC–ESI–MS/MS spectra of proto- A seventh surfactin isoform of LC–ESI–MS/MS spec- nated ions found at m/z 994 at retention time 6.38 min trum of species observed as protonated ion was showed 1 2 3 (Additional file  1: Figure S2a), 1008 at retention time in Fig. 5e with a peptide sequence of Glu -Leu/Ile -Leu - 4 5 6 7 6.94 min (Additional file  1: Figure S2b), 1036 at reten- Leu/Ile -Asp -Leu -Leu/Ile and a C β-hydroxyl fatty tion time 8.63  min (Additional file  1: Figure S2c) and acid chain with no Val. 1050 at retention time 9.63 min (Additional file 1: Fig- Figure  6 displayed the LC–ESI–MS/MS spectrum of ure S2d) with multiples of 14  Da (−CH2) difference, the precursor ion at m/z 1050 at retention time 4.68 min. were proved to be homologs with the same amino acid It could be concluded that it was a kind of esperin with 2 3 4 sequence but different C , C , C and C β-OH fatty the sequence could be surmised as Val -Leu - Leu/Ile - 12 13 15 16 5 6 7 acids. Asp -Leu -Leu/Ile with a C β-OH fatty acid based on Figure  4c and Additional file  1: Figure S3 showed a its chromatographic behavior and fragmentation pattern. third surfactin isoforms of a series of homologs. Fig- In the same way, we can conclude that the sequence of ure  4c showed the LC–ESI–MS/MS spectrum of spe- the precursor ion at m/z 1050 at retention time 5.12 min 1 2 3 4 cies observed as protonated ion at m/z 1022 at retention was C β-OH fatty acid-Glu -Leu/Ile -Leu -Leu/Ile - 5 6 7 time 8.11  min. Which was suggested the precursor ion Asp -Leu -Val (Additional file  1: Figure S4), an esperin, 1 2 3 possessed a peptide sequence of Glu -Val -Leu -Leu/ too. 4 5 6 7 Ile -Asp -Leu -Leu/Ile and a C β-hydroxyl fatty acid As it is more, Fig.  7a showed the LC–ESI–MS/MS chain. Simultaneously, the LC–ESI–MS/MS spectra spectrum of species observed as protonated ion at m/z of protonated ions found at m/z 994 at retention time 1026 at retention time 5.26 min. We can conclude it was 6.42  min (Additional file  1: Figure S3a), 1008 at reten- a linear surfactin with a peptide sequence of Glu -Leu/ 2 3 4 5 6 7 tion time 6.89  min (Additional file  1: Figure S3b), 1036 Ile -Leu -Val -Asp -Leu -Leu/Ile and a C β-hydroxyl at retention time 8.69  min (Additional file  1: Figure fatty acid chain. In addition, the protonated ion m/z at S3c) and with multiples of 14  Da (−CH ) difference, 1012 at retention time 4.79 min (Additional file  1: Figure were proved to be homologs with the same amino acid S5a), 1040 at retention time 5.89  min (Additional file  1: sequence but different C , C and C β-OH fatty Figure S5b), 1054 at retention time 6.36 min (Additional 12 13 15 acids. file  1: Figure S5c) and 1068 at retention time 7.19  min The fourth surfactin isoforms of LC–ESI–MS/MS (Additional file  1: Figure S5d) were assigned as homologs spectrum of species observed as protonated ion were with C , C , C , C β-OH fatty acid. Using the same 11 13 14 15 showed in Fig.  4d, e. Figure  4d showed fragment ions method, we can easily identify the protonated ion at m/z of precursor ion m/z 1008 at retention time 8.40  min. 1026 at the retention time 5.53  min was a linear surfac- 1 2 3 Then the sequence could be concluded based on the tin possessing a peptide sequence of Glu -Leu/Ile -Leu - 1 4 5 6 7 fragmentation profile as C β-OH fatty acid—Glu - Val -Asp -Leu -Val and a C β-hydroxyl fatty acid chain 14 14 2 3 4 5 6 7 Leu/Ile -Leu -Val -Asp -Leu -Val . In addition to this, (Fig. 7b). a protonated ion m/z at 1022 at retention time 9.35 min The protonated ion species of surfactin family con - was assigned as homologs with C β-OH fatty acid taining with surfactins, esperins and linear surfactins (Fig. 4e). isoforms characterized from the culture supernatants of Figure  5b showed the LC–ESI–MS/MS spectrum of marine B. megatherium are summarized in Table 1. species observed as protonated ion at m/z 1022 at reten- tion time 8.11 min. Then the sequence could be surmised LC–ESI–MS/MS characterisation of iturin lipopeptides 1 2 3 4 5 6 as C β-OH fatty acid-Glu -Val -Leu -Val -Asp -Leu - Figure  8 displayed the LC–ESI–MS/MS spectrum of the Leu/Ile . Thus, a protonated ion m/z at 1008 at retention precursor ion at m/z 1031 with appearance of fragment time 8.46 min was assigned as homologs with C β-OH ions at retention time 6.98  min. A series y fragment at 7 6 5 fatty acid (Fig. 5a). m/z 317 (+H O, 335)(Thr -Ser -Glu ),528 (−H O, 510) 2 2 7 6 5 4 3 7 6 In the same way, we inferred the sequence of the pre- (Thr -Ser -Glu -Pro -Asn ), 691 (−H O, 673)(Thr -Ser - 5 4 3 2 1 7 6 5 cursor ion at m/z 1008 at retention time 8.40  min and Glu -Pro -Asn -Tyr -Asn ) and 805 (Thr -Ser -Glu - 4 3 2 1 1022 at retention time 9.16  min were C β-OH fatty Pro -Asn -Tyr -Asn -β-amino fatty acid) were found in 1 2 3 4 5 6 7 + acid-Glu -Val -Leu -Leu/Ile -Asp -Leu -Val and C the MS spectrum of m/z 1031. The b fragment ion at 15 2 Ma et al. AMB Expr (2016) 6:79 Page 7 of 15 ab 671.30 671.21 90 90 990.66 80 80 60 60 649.30 877.96 1004.46 50 50 782.67 40 568.36 796.35 582.22 441.14 909.37 441.02 455.35 469.04 891.49 20 20 554.27 423.24 554.19 10 10 370.26 917.25 450 500 550 600 650 700 750 800 850 900 950 1000 400 500 600 700 800 900 1000 m/z m/z cd 671.29 671.38 100 681.53 441.15 990.52 653.61 554.26 40 1004.57 909.36 568.25 30 451.22 923.41 695.53 810.46 796.51 20 582.29 469.26 356.28 352.19 792.25 699.74 300 400 500 600 700 800 900 1000 400 500 600 700 800 900 1000 m/z m/z 990.51 568.19 50 681.16 536.36 30 423.38 782.61 328.06 554.13 895.34 441.24 20 667.24 342.13 455.20 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z Fig. 5 LC-ESI–MS/MS spectrum of the surfactin precursors. a, b Surfactin precursors ion [M + H] at m/z 1008 at retention time 8.46 and 9.22 min containing a Glu1-Val2-Leu3-Val4-Asp5-Leu6-Leu/Ile7 peptide and a C14 and C15 β-hydroxy fatty acid chain, respectively. c, d Surfactin precursors ion [M + H] at m/z 1008 at retention time 8.40 and 9.16 min containing a Glu1-Val2-Leu3-Leu/Ile4-Asp5-Leu6-Val7 peptide and a C14 and C15 β-hydroxy fatty acid chain, respectively. e Surfactin precursor ions ion [M + H] at m/z 1008 at retention time 6.89 min containing a Glu1-Leu/Ile2- Leu3-Leu/Ile4-Asp5-Leu6-Leu/Ile7 peptide and a C12 β-hydroxy fatty acid chain Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 8 of 15 The protonated ion species of bacillomycin D homologs 806.51 characterized from the culture supernatants of marine B. megaterium are summarized in Table 1. 708.53 LC/ESI–MS/MS characterisation of fengycin lipopeptides 937.55 Figure  9a displayed the LC–ESI–MS/MS spectrum of 30 the precursor ion at m/z 1479 with appearance of frag- 464.29 ment ions at retention time 12.63  min. The results 595.42 10 365.44 482.31 1032.71 919.36 + showed the appearance of b product ions at m/z 300 400 500 600 700 800 900 1000 1100 1200 1479  →  1177(−H O, 1159)  →  1080  →  952  →  676(− m/z 4 5 H O, 658) were assigned as the losses of (Thr -Glu - 6 7 8 9 10 + Ala )-Pro -Gln -(Tyr -Ile ) and the y product ions at m/z 1210 → 1081 → 967 were corresponded to the neu- 1 2 + tral losses of side-chain Glu –Orn . These fragment ions Fig. 6 LC-ESI–MS/MS spectrum of esperin precursors ion [M + H] at 3 4 were formed upon cleavage at Tyr and Thr in the mid- m/z 1050 at retention time 4.68 min containing a Glu1-Leu/Ile2-Leu3- Leu/Ile4-Asp5-Leu6-Val7 peptide and a C16 β-hydroxy fatty acid chain dle of molecule. A third series fragment ions at m/z 1408 6 5 6 and 1279 meant the losses of Ala and Glu - Ala , respec- tively. This fragment series suggested there was another 6 7 1 2 3 ring cleavage site between Ala and Pro of fengycin A m/z 617 (β-amino fatty acid-Asn -Tyr -Asn ) and 948 1 2 3 4 5 6 homolog. (β-amino fatty acid-Asn -Tyr -Asn -Pro -Glu -Ser ) were Figure  9b showed the LC–ESI–MS/MS spectrum of found, too. According to these typical CID fragments, 2+ the precursor ion [M  +  2H] at m/z 740 with appear- the sequence could be deduced as C β-amino fatty acid 1 2 3 4 5 6 7 ance of fragment ions at retention time 12.69  min. Dis- chain-Asn -Tyr -Asn -Pro -Glu -Ser -Thr , which was sociation yielded b ions at m/z 1479  →  1177  →  1080  assigned as bacillomycin D homologs. →  952  →  789  →  676 were surmised as the losses of The MS/MS spectrum of other protonated precursor 4 5 6 7 8 9 10 + (Thr -Glu -Ala )-Pro -Gln -Tyr -Ile and y ions at m/z ions at m/z 1045 at retention time 7.95  min (Additional 1081 and 967 were corresponded to the neutral loses of file  1: Figure S6) also yielded similar profile of product β-hydroxyl fatty acid chain-Glu and β-hydroxyl fatty ions was proved to be homologs possessing the same 1 2 acid chain-Glu -Orn , respectively. From the product amino acid sequence but different C β-amino fatty + 2+ ions, [M + H] at m/z 1479 and [M + 2H] at m/z 734 acids. ab x10 2.8 2.6 2 x10 568.5 2.4 1.1 671.6 2.2 356.3 455.2 0.9 685.3 1.8 0.8 667.2 1.6 0.7 1.4 0.6 469.4 342.0 1.2 0.5 582.1 0.4 441.2 0.3 0.8 451.2 227.0 328.1 0.2 0.6 681.5 778.0 0.1 0.4 554.3 227.4 0.2 199.3 -0.1 100 200 300 400 500 600 700 800 900 150 200 250 300 350 400 450 500 550 600 650 700 750 800 m/z m/z Fig. 7 LC-ESI–MS/MS spectrum of the linear surfactin precursors. a Linear surfactin precursors ion [M + H] at m/z 1026 at retention time 5.26 min containing a Glu1-Leu/Ile2-Leu3-Val4-Asp5-Leu6-Leu/Ile7 peptide and a C13 β-hydroxy fatty acid chain. b Linear surfactin precursor ions [M + H] at m/z 1026 at retention time 5.53 min containing a Glu1-Leu/Ile2-Leu3-Val4-Asp5-Leu6-Val7 peptide and a C14 β-hydroxy fatty acid chain Intensity Relative Abundance(%) Intensity Ma et al. AMB Expr (2016) 6:79 Page 9 of 15 Table 1 Assignment of the structures of lipopeptides by LC–ESI–MS/MS in this study Peak Mass (M/S) Rt (min) Family Assignment Sequence 1 2 3 4 5 6 7 1 994 6.50 Surfactin C [M + H] 12 R -CH-CH -CO-Glu -Leu/Ile -Leu -Val -Asp -Leu -Leu/Ile n 2 2 1008 6.98 C [M + H] 3 1022 8.05 C [M + H] 4 1036 9.12 C [M + H] 5 1050 9.38 C [M + H] 1 2 3 4 5 6 7 6 994 6.38 C [M + H] 12 R -CH-CH -CO-Glu -Leu/Ile -Leu -Leu/Ile -Asp -Leu -Val n 2 7 1008 6.94 C [M + H] 8 1022 8.17 C [M + H] 9 1036 8.63 C [M + H] 10 1050 9.63 C [M + H] 1 2 3 4 5 6 7 11 994 6.42 C [M + H] 12 R -CH-CH -CO-Glu -Val -Leu -Leu/Ile -Asp -Leu -Leu/Ile n 2 12 1008 6.89 C [M + H] 13 1022 8.11 C [M + H] 14 1036 8.69 C [M + H] 1 2 3 4 5 6 7 16 1008 8.40 C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Val -Asp -Leu -Val 14 n 2 17 1022 9.35 C [M + H] 1 2 3 4 5 6 7 18 1008 8.46 C [M + H] 14 R -CH-CH -CO-Glu -Val -Leu -Val -Asp -Leu -Leu/Ile n 2 19 1022 9.22 C [M + H] 1 2 3 4 5 6 7 20 1008 8.40 Surfactin C [M + H] R -CH-CH -CO-Glu -Val -Leu -Leu/Ile -Asp -Leu -Val 14 n 2 21 1022 9.16 C [M + H] 1 2 3 4 5 6 7 22 1008 6.89 C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Leu/Ile -Asp -Leu -Leu/Ile 12 n 2 1 2 3 4 5 6 7 23 1050 4.68 Esperin C [M + H] 16 R -CH-CH -CO-Glu -Val -Leu -Leu/Ile -Asp -Leu -Leu/Ile -COOH n 2 1 2 3 4 5 6 7 24 1050 5.12 C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Leu/Ile -Asp -Leu -Val -COOH 16 n 2 1 2 3 4 5 7 25 1012 4.81 Surfactin Linear C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Val -Asp -Leu6-Leu/Ile -COOH n 2 OH 26 1026 5.26 Linear C [M + H] 27 1040 5.87 Linear C [M + H] 28 1054 6.39 Linear C [M + H] 29 1068 7.17 Linear C [M + H] 1 2 3 4 5 6 7 30 1026 5.53 Surfactin Linear C [M + H] R -CH-CH -CO-Glu -Leu/Ile -Leu -Val -Asp -Leu -Val -COOH 14 n 2 OH Ma et al. AMB Expr (2016) 6:79 Page 10 of 15 Table 1 continued Peak Mass (M/S) Rt (min) Family Assignment Sequence 1 2 3 4 5 6 7 31 1031 6.98 Bacillomycin D C [M + H] 14 R -CH-CH -CO-Asn -Tyr -Asn -pro -Glu -Ser -Thr n 2 NH 32 1045 7.95 C [M + H] 1 2 3 4 5 6 33 1451 10.94 Fengycin A C [M + H] 15 R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Ala n 2 10 9 8 7 OH Ile -Tyr -Gln -Pro 34 1465 10.82 C [M + H] 35 1479 12.63 C [M + H] 2+ 36 740 12.69 C [M + 2H] 37 1493 13.01 C [M + H] 2+ 38 747 15.09 C [M + 2H] 1 2 3 4 5 6 39 1479 12.71 Fengycin B C [M + H] 15 R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Val n 2 10 9 8 7 OH Ile -Tyr -Gln -Pro 2+ 40 740 12.61 C [M + 2H] 41 1493 12.92 C [M + H] 2+ 42 747 13.15 C [M + 2H] 1 2 3 4 5 6 7 8 9 10 43 1467 8.66 Fengycin A Linear C [M + H] R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Ala -Pro -Gln -Tyr -Ile -COOH 15 n 2 OH 44 1481 8.83 Linear C [M + H] 45 1495 12.36 Linear C [M + H] 1 2 3 4 5 6 7 8 9 10 46 1495 12.36 Fengycin B Linear C [M + H] 15 R -CH-CH -CO-Glu -Orn -Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile -COOH n 2 OH 47 1509 13.87 Linear C [M + H] were with the same structure, it could be concluded that 1013.50 1 2 it was a kind of fengycin A with the sequence Glu –Orn - 3 4 5 6 7 8 9 10 805.09 Tyr -Thr -Glu -Ala -Pro -Gln -Tyr -Ile with a C β- 335.01 OH fatty acid based on its chromatographic behavior and 60 673.36 fragmentation pattern. 492.40 In addition, the protonated ion m/z at 1451 at reten- 617.16 510.82 30 599.26 tion time 10.94  min (Additional file  1: Figure S7a), 1465 948.55 at retention time 10.82  min (Additional file  1: Figure S7b), 1493 at retention time 13.01 min (Additional file  1: 400 500 600 700 800 900 1000 2+ Figure S7c) and [M + 2H] at m/z 747 at retention time m/z 15.09  min (Additional file  1: Figure S7d) with multiples of 14  Da difference in their molecular ion species were proved to be Fengycin A homologs with the same amino acid sequence but different C , C , C and C β-OH 15 16 18 18 fatty acids chain. Fig. 8 LC-ESI–MS/MS spectrum of the bacillomycin D precursors ion [M + H] at m/z 1031 at retention time 6.98 min containing a A second fengycin isoforms of LC–ESI–MS/MS spec- Asn1-Tyr2-Asn3-pro4-Glu5-Ser6-Thr7 peptide and a C14 β-amino fatty trum of species observed as protonated ion were showed acid chain on Fig.  9c, d and Additional file  1: Figure S8. Then the Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 11 of 15 ab 967.39 1080.95 952.38 40 1081.71 1408.24 30 676.53 789.49 952.44 1279.58 1159.21 658.40 20 1081.49 967.67 1177.58 1210.83 1080.50 1279.02 500 600 700 800 900 1000 1100 1200 1300 1400 400 500 600 700 800 900 1000 1100 1200 m/z m/z cd 995.48 1109.28 1109.65 991.57 814.37 775.43 1216.16 662.29 1145.60 832.39 731.33 502.56 20 389.13 20 226.44 1238.78 775.59 1163.62 1048.74 938.44 832.39 938.44 644.66 749.27 995.61 1145.62 385.25 10 499.32 10 895.06 503.21 500 600 700 800 900 1000 1100 1200 1300 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z m/z Fig. 9 LC-ESI–MS/MS spectrum of the fengycin precursors. a Fengycin A precursors ion [M + H] at m/z 1479 at retention time 12.63 min contain- ing a Glu1-Orn2-Tyr3-Thr4-Glu5-Ala6-Pro7-Gln8-Tyr9-Ile10 peptide and a C17 β-hydroxy fatty acid chain. b Fengycin A precursors ion [M + 2H]2 at m/z 740 at retention time 12.69 min containing a C17 β-hydroxy fatty acid chain. c Fengycin B precursors ion [M + H] at m/z 1493 at retention time 12.92 min containing a Glu1-Orn2-Tyr3-Thr4-Glu5-Val6-Pro7-Gln8-Tyr9-Ile10 peptide and a C16 β-hydroxy fatty acid chain. d Fengycin B precursors 2+ ion [M + 2H] at m/z 747 at retention time 13.15 min containing a C16 β-hydroxy fatty acid chain 2+ sequence of Fig.  9c could be concluded based on the Figure S8a) and [M + 2H] at m/z 740 at retention time 1 2 fragmentation profile as C β-OH fatty acid-Glu –Orn - 12.61 min (Additional file  1: Figure S8b) were assigned as 3 4 5 6 7 8 9 10 Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile . Figure  9d fengycin B homologs with C β-OH fatty acid. 2+ showed fragment ions of precursor ion [M  +  2H] at Figure  10a showed the LC–ESI–MS/MS spectrum of m/z 747 at retention time 13.15  min. It turned out that species observed as protonated ion at m/z 1495 at reten- it has the same structure with m/z 1493 at retention tion time 12.36 min. We concluded it is a linear fengycin 1 2 3 4 5 time 12.92 min. In addition to this, a protonated ion m/z A with a peptide sequence of Glu -Orn -Tyr -Thr -Glu - 6 7 8 9 10 at 1479 at retention time 12.71  min (Additional file  1: Ala -Pro -Gln -Tyr -Ile and a C β-hydroxyl fatty acid Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 12 of 15 a b 1013.22 493.19 40 40 922.17 521.21 1054.19 30 975.30 1363.63 1127.41 747.41 656.38 20 975.30 1200.57 20 1200.57 1363.63 775.24 1381.49 876.46 967.24 483.45 1054.19 592.38 721.45 10 1381.49 500 600 700 800 900 1000 1100 1200 1300 1400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z m/z Fig. 10 LC-ESI–MS/MS spectrum of the linear fengycin precursors. a Linear fengycin A precursors ion [M + H] at m/z 1495 containing a Glu1- Orn2-Tyr3-Thr4-Glu5-Ala6-Pro7-Gln8-Tyr9-Ile10 peptide and a C17 β-hydroxy fatty acid chain with a double bond. b Linear fengycin B precursors ion [M + H] at m/z 1495 containing a Glu1-Orn2-Tyr3-Thr4-Glu5-Val6-Pro7-Gln8-Tyr9-Ile10 peptide and a C15 β-hydroxy fatty acid chain with a double bond chain with a double bond based on its chromatographic families of molecules were successively separated and behavior and fragmentation pattern. The protonated ion characterised by LC–ESI–MS in positive full scan mode. m/z at 1467 at retention time 8.66 min (Additional file  1: Surfactin family contains more than 20 different lipo - Figure S9a) and 1481 at retention time 8.79  min (Addi- peptides such as lichenysin, pumilacidin, esperin and tional file  1: Figure S9b) with characteristic fragment ions surfactin. Esperin is coincided with those established sur- m/z 967 and 1081 were assigned as linear fengycin A factin for the composition and the amino acid residues, homologs with C and C β-OH fatty acid with a double except that there is a smaller peptide ring formed by the 15 16 bond, respectively. fifth l-Asp in the esperin, and not by the seventh Leu- The LC–ESI–MS/MS spectrum of species observed residue like surfactin (Kalinovskaya et  al. 1995; Thomas as protonated ion at m/z 1495 at retention time and Ito 1969). Surfactins have the common following 12.36  min had a second series of fragmentation ions structural traits: a heptapeptide with a chiral sequence were showed that it contained a peptide sequence of LLDLLDL interlinked with a β-hydroxy fatty acid and 1 2 3 4 5 6 7 8 9 10 Glu -Orn -Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile with a d-Leu in position 3 and 6 and a l-Asp in position and a C β-hydroxyl fatty acid chain with a double 5. Amino acid residues in position 2, 4 and 7 belong to bond (Fig.  10b). Besides, a protonated ion m/z at 1509 the amino acids group including Val, Leu and Ile (Jacques at retention time 13.87  min with characteristic frag- 2011; Hue et  al. 2001). With a Gln in the position 1 are ment ions m/z 995 was assigned as linear fengycin B named lichenysin which are different from surfactin with homologs with C β-OH fatty acid with a double bond the Glu in the position 1 (Qiu et  al. 2014). Some of the (Additional file 1 : Figure S10). isoforms had the same m/z by ESI–MS, but they were in The protonated ion species of fengycin family con - different HPLC fraction. Thus, it is necessary to precisely taining with fengycin A, B and linear fengycin isoforms identify their structure by MS/MS, especially for the characterized from the culture supernatants of marine B. amino acid sequence of the peptide portion of the mol- megaterium are summarized in Table 1. ecules (Yang et al. 2015). According to the results of LC–MS, ten major frac- Discussion tions with different molecular weight were used as pre - LC–ESI–MS was performed in order to investigate the cursor ions for further ESI–MS/MS analysis. In details, nature of the compounds in the methanol extract after the precursor ions at m/z 994, 1008, 1022, 1036 and 1050 a two-step ultrafiltration. Different families of lipopep - were hypothesized to be a series of homologue molecules tides with surfactins, fengycins and iturins can be eluted of surfactins with 14 or multiples of 14  Da difference in by 85–100, 50–70, 40–50  % acetonitrile in water, respec- their molecular ion species. tively, in reversed phase high performance liquid chroma- From the results of LC–ESI–MS/MS of Figs.  4 and 5, tography (RP-HPLC) system (Yang et al. 2015). The three the presence of fragment ions at m/z 685, 671 or 681 are Relative Abundance(%) Relative Abundance(%) Ma et al. AMB Expr (2016) 6:79 Page 13 of 15 the common peaks in CID spectrum of surfactin isoforms (Maget-Dana and Peypoux 1994). Bacillomycin D, closely ion. The amino acids sequences at m/z 671, 685 and 681 related variants with iturin, is famous for the high activity 2 3 4 5 6 7 + are [(H)Leu/Ile -Leu -Val -Asp -Leu -Val (OH)  +  H] / against Aspergillus flavus (Moyne et al. 2001). According 2 3 4 5 6 7 + [(H)Val -Leu -Val -Asp -Leu -Leu/Ile (OH)  +  H] /[(H) to the results of LC–MS, the most intense fractions with 2 3 4 5 6 7 + Val -Leu -Leu/Ile -Asp -Leu -Val (OH) +  H] , [(H)Leu/ different molecules at m/z 1031 and 1045 were used as 2 3 4 5 6 7 + 2 Ile -Leu -Val -Asp -Leu - Leu/Ile (OH)  +  H] /[(H)Val - precursor ions by ESI–MS/MS analyzes for further pep- 3 4 5 6 7 + + Leu -Leu/Ile -Asp -Leu -Leu/Ile (OH)  +  H] /[(H)Leu/ tide sequence determination and some others [M +  H] 2 3 4 5 6 7 + Ile -Leu -Leu/Ile -Asp -Leu -Val (OH)  +  H] and [(H) at m/z 1059 and 1073. They were hypothesized to be a 2 3 4 5 6 7 + Leu/Ile -Leu -Leu/Ile -Asp -Leu -Leu/Ile (OH)  +  H] , series of homologous molecules of bacillomycin D with respectively. u Th s, the fragment ions at m/z 685, 671 or multiples of 14  Da difference in their molecular ion 681 are characteristic marker ions for identification of species. surfactins. Fengycin homologs are divided into two different types Through LC–ESI–MS, the chromatogram for surfactin (fengycin A and B) by their amino acid sequence. The family showed two unique peaks at retention time 4.68 fengycin A has the Ala at position 6, while Val for fengy- + 9 and 5.12 min corresponding to molecules [M +  H] m/z cin B (Vater et  al. 2002). Fengycins (plipastatin if Tyr is 1050. Their retention times were much less than the sur - D-configured) (Raaijmakers et  al. 2010), the configura - factin standard time, thus, according to the principle of tion of amino acid sequence is LDLDLDDLLDL with a similar miscibility, it showed that these molecules with lactone bond connecting l-Tyr to l Ile, includes fengy- stronger polarity. Figure 6 showed the dissociation yielded cins A, B and relevant homologs with the difference b ions at m/z 1050 (−H O, 1032) → 919 → 806 → 708  in the amino acid at position 6. They are macrolactone →  595  →  482  →  383 (−H O, 365) were surmised as rings with the Tyr side chain at position 3 of the peptide 7 6 5 4 the losses of (OH)Leu/Ile -Leu -Asp(-OH) -Leu/Ile - sequence and the C-terminal residue yielding an internal 3 2 Leu -Val from the C terminus. Therefore, we can infer ring by an ester bond. 1 2 3 4 5 its amino acids sequence is Glu -Val -Leu -Leu/Ile -Asp - From the above, the results (Fig. 9a, b; Additional file  1: 6 7 Leu -Leu/Ile , which is the same with surfactin. But, its Figure S7) showed the appearance of product ions at m/z fragment ions didn’t have the characteristic fragment ion 1081 and 967 from a series of precursor ions and the m/z 685 (Figs.  4, 5) which can be inferred it wasn’t sur- results (Fig. 9c, d and Additional file  1: Figure S8) showed factin. Simultaneously, the peak from m/z 806 to 708 in the appearance of product ions at m/z 1109 and 995 from Fig.  6 was formed by loss of the Asp(−OH) from mol- a series of precursor ions. The molecular weights of two ecule, which confirmed that the carboxyl group of Asp group peaks (1081 and 1109, 967 and 995) were differed and the hydroxyl group of the aliphatic part formed the by 28 Da, suggesting a difference in the amino acid com - ester structure in the esperin molecule, and this is another position of the peptide (Ala or Val). Thus, m/z 1080 and evidence of our inferences. The most noteworthy is that 967 are characteristic ions for fengycin A and m/z 1109 the fragment ions of it at m/z 450 were matched with the and 995 are characteristic ions for fengycin B with the + 7 6 5 losses of H (OH)Val -Leu -Asp -C β-OH fatty acid information of specific octapeptide ring ions. from the C terminus which further improved the carboxyl The result (Fig.  10a) showed the b fragment ions at group of Asp and the hydroxyl group of the aliphatic part m/z 1495(−H O, 1477)  →  1363  →  1200  →  1072(-H O, 2 2 formed the ester structure in the esperin molecule. 1054) →  975 →  904(+H O, 922) →  775 →  674(−H O, 2 2 According to LC–ESI–MS, before the elution of sur- 656) → 511(−H O, 493) matched with the losses of Tyr - 4 5 6 7 8 9 10 + factins (4.5–7.2 min), several peaks appeared that corre- Thr -Glu -Ala -Pro -Gln -Tyr -Ile . Its y fragment ions spond to unknown compounds at m/z 1012 at retention at m/z 985(−H O, 967) → 721 → 592 → 521 meant the 3 4 5 6 time 4.79  min, 1026 at retention time 5.26  min, 1026 at losses of (Tyr -Thr )-Glu -Ala in the middle of precursor retention time 5.53 min, 1040 at retention time 5.89 min, ion and the peak of 967 is the characteristic fragment ion 1054 at retention time 6.36  min and 1068 at retention of fengycin A. It was 16 Da bigger than m/z 1479. We con- time 7.19  min. What a coincidence is that, compared cluded it was a linear fengycin A with a peptide sequence 1 2 3 4 5 6 7 8 9 10 with the surfactins at m/z 994, 1008, 1022, 1036 and of Glu -Orn -Tyr -Thr -Glu -Ala -Pro -Gln -Tyr -Ile 1050, respectively, the m/z precursor molecules are 18 Da and a C β-hydroxyl fatty acid chain with a double bond. bigger than surfactins correspondingly. As we know, the The result (Fig.  10b) showed the b fragment ions at m/z relative molecular mass of the H O was just 18. We can 1495(−H O, 1477)  →  1363  →  1200  →  1072(−H O, 2 2 2 conclude they were linear surfactins based on their chro- 1054) → 975 → 876 → 747 → 483 revealed the losses of 3 4 5 6 7 8 9 10 matographic behavior and fragmentation pattern. Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile from the C ter- Though iturin has the limited antiviral and antibac - minus. Its y fragment ions at m/z 1127 → 1013 meaned terial activity, but it displays strong antifungal activity the losses of Orn , suggesting the precursor ion (a linear Ma et al. AMB Expr (2016) 6:79 Page 14 of 15 1 2 Acknowledgements fengycin B) possessed a peptide sequence of Glu -Orn - 3 4 5 6 7 8 9 10 Dr. Yejun Han from Institute of Process Engineering, Chinese Academy of Sci- Tyr -Thr -Glu -Val -Pro -Gln -Tyr -Ile and a C ences, is appreciated for helping improve this manuscript. β-hydroxyl fatty acid chain with a double bond. Competing interests LC–ESI–MS/MS was performed after a two-step The authors declare that they have no competing interests. ultrafiltration in order to investigate the nature of the compounds in the methanol extract of marine B. mega- Ethics approval and consent to participate This article does not contain any studies concerned with experimentation on terium. The three families of molecules were successively human or animals. separated and characterised by LC–ESI–MS in positive full scan mode. LC–ESI–MS/MS analysis precisely eluci- Funding This study was funded by the National Natural Science Foundation of China dated the different amino sequence of isoforms of iturin, (No. 31471657). surfactin and fengycin families. Collectively, we have established seven kinds of cyclic isoforms of surfactin Received: 7 September 2016 Accepted: 8 September 2016 with a series of homologs (Table 1). 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Published: Sep 17, 2016

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