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

Abstract

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

Screening of a <i>Bacillus subtilis</i> Strain Producing Multiple Types of Cyclic Lipopeptides and Evaluation of Their Surface-tension-lowering Activities

“…By contrast, the spectra of CLP-II exhibited the major M at m/z 1073 and 1043. However, the spectra of CLP-II were different from the data that were previously reported for fengycin 32 , and the compounds could not be identified based on the MALDI-TOF-MS analysis alone. According to the results of the PCR, strain NBRC 109107 should produce the fengycin homologues.…”

Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review

“…One of the simplest methods for the recovery of LP is acid precipitation. [104] Acid precipitation is carried out using concentrated HCl to lower the pH to 2.0, which neutralizes negative charges on LP, making them less soluble in the aqueous phase. The precipitated LP can then be collected for analysis.…”

Fengycins, Cyclic Lipopeptides from Marine Bacillus subtilis Strains, Kill the Plant-Pathogenic Fungus Magnaporthe grisea by Inducing Reactive Oxygen Species Production and Chromatin Condensation

“…CLPs from Bacillus species can be divided into three main subfamilies, surfactins, iturins, and fengycins or plipastatins. Among these CLPs, fengycins exhibit powerful antifungal activity against a wide range of phytopathogens (9). Fengycin homologues effectively inhibit the growth of filamentous fungi, such as Rhizopus stolonifer, Gibberella zeae, Aspergillus niger, Mucor rouxii, Fusarium graminearum, and Sclerotinia sclerotiorum (10–13).…”