Neutral Loss Fragmentation Pattern Based Screening for Arginine-Rich Natural Products in Xenorhabdus and Photorhabdus

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-MS(2), and a database comprising MS(2) 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.

Amine-reactive Neutron-encoded Labels for Highly Plexed Proteomic Quantitation

“…Second, the label is relatively large; ϳ435 Da is added to each primary amine (i.e., one to two per peptide). Third, Arg is the most basic amino acid and may inhibit random backbone fragmentation by sequestering protons (46,47). The reduction in proteomic coverage observed in the 12-plex experiments, as compared with the 4-plex data, is likely caused by increased spectral complexity; that is, three isotopic clusters are observed for every unique peptide sequence, so that lower abundance species are less often selected for MS/MS (45,48).…”

ω-Azido fatty acids as probes to detect fatty acid biosynthesis, degradation, and modification

“…Silica chromatographic purifi cation was performed on a Biotage SP1™ Flash purifi cation system (Biotage, Uppsala, Sweden), using 40+M, 25+M, or 12+M KP-Sil cartridges (Biotage, Uppsala, Sweden) in combination with an UV detector. C-HMBC NMR spectra for the synthesis products were recorded on a Bruker AV500 (500 MHz), AV400 (400 MHz), or AM250 (250 MHz) spectrometer using CDCl 3 or CD 3 CN as solvent and internal standard, using the chemical shifts described by Gottlieb et al ( 11 ) with the exception of the 13 Cshift of CDCl 3 , which was set to 77.00 ppm. were also detectable when high concentrations of the corresponding nonoxidized AFAs were reached (supplementary Fig.…”

“…High resolution mass spectra were obtained from a MALDI LTQ Orbitrap XL (Thermo Fisher Scientifi c, Inc., Waltham, MA) equipped with a laser at 337 nm. A 4-chloro-␣ -cyanocinnamic acid matrix was used, and the sum formulas and according masses were internally calibrated using fl uorescein (monoisotopic mass = 332.068473) as a standard ( 13 ).…”

Collision-Induced Dissociation Mass Spectrometry: A Powerful Tool for Natural Product Structure Elucidation

“…[20][21][22] Still, the full elucidation of novel structures frequently relies upon manual data interpretation and corresponding NMR-based assessment of these complex compounds, which are often produced at remarkably low physiological concentrations. Most reports of novel natural products use mass spectrometry as a confirmatory tool to NMR data; 23 however, the throughput of mass spectrometry warrants increased reliance as a structure elucidation tool. Additionally, mass spectrometry facilitates characterization of compounds which cannot be purified, either due to degradation 24 or relative abundance.…”