Article pubs.acs.org/ac
Neutral Loss Fragmentation Pattern Based Screening for ArginineRich Natural Products in Xenorhabdus and Photorhabdus Sebastian W. Fuchs,† Christian C. Sachs,† Carsten Kegler,† Friederike I. Nollmann,† Michael Karas,‡ and Helge B. Bode*,† †
Merck Stiftungsprofessur für Molekulare Biotechnologie, Instititut für Molekulare Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany ‡ Cluster of Excellence Macromolecular Complexes, Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, 60438 Frankfurt, Germany S Supporting Information *
ABSTRACT: Although sharing a certain degree of structural uniformity, natural product classes exhibit variable functionalities such as different amino acid or acyl residues. During collision induced dissociation, some natural products exhibit a conserved fragmentation pattern close to the precursor ion. The observed fragments result from a shared set of neutral losses, creating a unique fragmentation pattern, which can be used as a fingerprint for members of these natural product classes. The culture supernatants of 69 strains of the entomopathogenic bacteria Photorhabdus and Xenorhabdus were analyzed by MALDI-MS2, and a database comprising MS2 data from each strain was established. This database was scanned for concordant fragmentation patterns of different compounds using a customized software, focusing on relative mass differences of the fragment ions to their precursor ion. A novel group of related natural products comprising 25 different arginine-rich peptides from 16 different strains was identified due to its characteristic neutral loss fragmentation pattern, and the structures of eight compounds were elucidated. Two biosynthesis gene clusters encoding nonribosomal peptide synthetases were identified, emphasizing the possibility to identify a group of structurally and biosynthetically related natural products based on their neutral loss fragmentation pattern.
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different amino acid or acyl residues as well as other modifications.5 Thus, an automated screening approach for the identification of novel members of a natural product family cannot only focus on mass and/or retention time. In the case of ribosomally or nonribosomally derived peptidyl natural products, such a screening could for example be based upon the search for shared amino acid-residue mass-shift series in tandem mass spectrometry (MS2) experiments as shown previously by Kersten et al.6 They used the identified shift series to characterize the corresponding peptide natural products and to link them to the genetic locus of their respective biosynthetic pathways. Another possibility would be the utilization of characteristic neutral loss- and/or fragmentions, the so-called neutral loss- and product ion scanning, for the identification of related natural products, as it is generally used for drug metabolite studies.7−9 Furthermore, the MS2 based neutral loss scanning is also commonly applied for the identification of phosphorylation or glycosylation sites in proteins10,11 and could also be used to identify natural products which are phosphorylated or glycosylated.
he complex life cycle of the entomopathogenic bacterial genera Xenorhabdus and Photorhabdus encompasses symbiotic interactions with their nematode hosts as well as pathogenicity toward the infected insect larvae.1,2 An additional requirement posed by the occupied ecological niche is the ability to defend the insect cadaver against pro- and eu-karyotic food competitors.3 These multiple interactions of entomopathogenic bacteria with their environment are partly governed by a variety of secreted secondary metabolites, exhibiting variable biological activities.3,4 During our search for novel natural products from these bacteria, we tried to identify natural products, which are present in several different strains. The rationale behind this was that the conserved production of a natural product or its derivatives in several different species would make this particular compound family more likely to be of ecological importance for the entomopathogenic bacteria and thus more probable to exhibit an interesting bioactivity. Since this survey requires the analysis of a large number of strains and compounds, an automated screening methodology had to be developed, which facilitates the identification of natural products without their time-consuming isolation. Although sharing a certain degree of structural uniformity, members of a family of related natural products, which we tried to identify in here, exhibit variable functionalities such as © 2012 American Chemical Society
Received: February 7, 2012 Accepted: July 23, 2012 Published: August 6, 2012 6948
dx.doi.org/10.1021/ac300372p | Anal. Chem. 2012, 84, 6948−6955
Analytical Chemistry
Article
investigated by feeding of L-[5,5,5-2H3]leucine (Sigma-Aldrich, ≥ 98+ % deuterium) to LB medium, where strains were handled as described above, except for the cultivation medium. Cultures were harvested 24 h after the last feeding by centrifugation at 12 000 rpm for 5 min. The supernatant was frozen at −20 °C. Sample Preparation and MALDI-MS as well as MALDIMS2. Culture supernatants were prepared for MALDI-analysis as a 1:6 dilution in 30% acetonitrile (ACN) with 0.1% trifluoracetic acid (TFA). All samples were mixed 1:2 with 1 μL of a 20 mM 4-chloro-α-cyanocinnamic acid (ClCCA)15,16 in 70% ACN and spotted onto a polished stainless steel target and air-dried. MALDI-MS and MALDI-MS2 analysis was performed with a MALDI LTQ Orbitrap XL (Thermo Fisher Scientific, Inc., Waltham, MA) equipped with a nitrogen laser at 337 nm. The following instrument parameters were used: laser energy, 15 μJ; automatic gain control, on; auto spectrum filter, off; resolution, 100 000; plate motion, survey CPS. For calculation of sum formulas, mass spectra were internally calibrated using calibration mixture 2 (Applied Biosystems, Sequazyme peptide mass standards kit). CID-MS2 experiments were in part performed in FTMS mode using the following parameters: resolution, 7500; wide band activation, on; precursor width range, optimized for every single precursor with regard to complete isolation; normalized collision energy, 40; Act. Q., 0.25; Act. Time, 30.0. Additionally, some MS2 experiments were performed in HCD mode using the following parameters: Act. Type, HCD; normalized collision energy, 45; Act. Q., 0.25; Act. Time, 30.0. When MS/MS-experiments were measured in ITMS mode, the following parameters were applied: Scan rate, normal; Act. Type, CID; Scan Type, full. Spectra were analyzed, and possible sum formulas were calculated using Qual Browser (version 2.0.7; Thermo Fisher Scientific, Inc., Waltham, MA). Internally calibrated spectra for sum formula calculation were measured as triplicates. Derivatization with Acetylacetone. For improved sequence-ion generation of arginine containing compounds during CID-MS2, arginine residues were converted into N5(4,6-dimethyl-2-pyrimydinyl)-ornithine (pyo) residues using acetylacetone derivatization as described by Dikler et al.17 This resulted in a reduced gas phase basicity of arginine side chains, thus promoting the fragmentation at peptide bonds, resulting in a strong promotion of sequence ions. Briefly, sterile culture supernatants of producing strains were mixed 1:1 with acetylacetone and shaked at 900 rpm and 50 °C for five days, resulting in the partial formation of completely derivatized compounds according to the number of arginine residues per molecule. Compounds of interest were enriched using C18 solid phase extraction (SPE). Strata-C18E SPE-columns (Phenomenex, Germany) were conditioned according to the manufacturer and equilibrated with aqueous 0.1% TFA. Supernatants were mixed 1:1 with aqueous 0.1% TFA and loaded onto the cartridge. The compounds were eluted with 10% ACN/0.1% TFA, 40% ACN/0.1% TFA, and 99.9% ACN/ 0.1% TFA. After evaporation to dryness, samples were dissolved in 70% ACN/0.1% TFA and subjected to MALDI mass spectrometry. Screening for Conserved Neutral Loss Fragmentation Patterns. In order to find relatively similar tandem mass spectra, similar to the tandem mass spectrum of 1, a customized software in the Ruby programming language was developed. MALDI-CID-ITMS2 scans in Thermo-Finnigan .RAW format were converted with Thermo-Finnigan Xconvert software to a
Here, we describe a screening methodology for the identification of a peptidyl-natural product-family with limited backbone fragmentation (strongly reduced formation of series ions: a-, b-, c- and x-, y-, z-ions) based on its conserved neutral loss fragmentation pattern (NLFP). This screening approach uses a conserved pattern of neutral loss signals occurring in collision induced dissociation-MS2 experiments in a mass range close to the precursor ion (
