High-resolution Ion Cyclotron Resonance Research Articles

High metabolomic microdiversity within co-occurring isolates of the extremely halophilic bacterium Salinibacter ruber.

Salinibacter ruber is an extremely halophilic member of the Bacteroidetes that thrives in crystallizer ponds worldwide. Here, we have analyzed two sets of 22 and 35 co-occurring S. ruber strains, newly isolated respectively, from 100 microliters water samples from crystalizer ponds in Santa Pola and Mallorca, located in coastal and inland Mediterranean Spain and 350 km apart from each other. A set of old strains isolated from the same setting were included in the analysis. Genomic and taxonomy relatedness of the strains were analyzed by means of PFGE and MALDI-TOF, respectively, while their metabolomic potential was explored with high resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS). Overall our results show a phylogenetically very homogeneous species expressing a very diverse metabolomic pool. The combination of MALDI-TOF and PFGE provides, for the newly isolated strains, the same scenario presented by the previous studies of intra-specific diversity of S. ruber using a more restricted number of strains: the species seems to be very homogeneous at the ribosomal level while the genomic diversity encountered was rather high since no identical genome patterns could be retrieved from each of the samples. The high analytical mass resolution of ICR-FT/MS enabled the description of thousands of putative metabolites from which to date only few can be annotated in databases. Some metabolomic differences, mainly related to lipid metabolism and antibiotic-related compounds, provided enough specificity to delineate different clusters within the co-occurring strains. In addition, metabolomic differences were found between old and new strains isolated from the same ponds that could be related to extended exposure to laboratory conditions.

Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR

Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2g/L) glucose concentration. The yields were identical using media containing 15NH4Cl or 15NH4Cl in combination with all-13C–d-glucose allowing to produce homogenous soluble monomeric NKp30 in several formats needed for advanced NMR studies. Our optimized protocol now allows to produce routinely 10mg batches of these NKp30ex proteins per 1L of M9 production medium in four working days. The purity and identity of the produced proteins were checked by SDS–PAGE, MALDI MS peptide mapping, and high resolution ion cyclotron resonance MS. Analytical ultracentrifugation confirmed the monomeric status of the produced proteins. Long-term stability of the produced protein proved to be very good allowing its use for NMR studies using elevated temperatures. These studies should reveal further details of the interaction of NKp30 with several of its ligands including target cell surface proteins and heparin-derived oligosaccharides.

Fine Structure in Isotopic Peak Distributions Measured Using a Dynamically Harmonized Fourier Transform Ion Cyclotron Resonance Cell at 7 T

The fine structure of isotopic peak clusters in mass spectra of reserpine and substance P are measured using Fourier transform ion cyclotron resonance mass spectrometry at a 7 T magnetic field. The resolved peaks in the fine structure consist of (13)C, (15)N, (17)O, (18)O, (2)H, (33)S, (34)S, and combinations of them. A recently introduced high-resolution ion cyclotron resonance cell (Nikolaev, E. N.; Boldin, I. A.; Jertz, R.; Baykut, G. J. Am. Soc. Mass Spectrom. 2011, 22, 1125-1133) is used in these experiments. The positions of the experimentally obtained fine structure peaks on the mass scale agree with the isotopic distribution simulations with ≤200 ppb error. Some deviation from the theoretical isotopic distribution is observed, less abundant peaks in the fine structure patterns are a little suppressed compared to the larger ones. We present a method for atomic composition determination using accurate mass data and fine isotopic structure of the mass spectrum. Our method combines the traditional atomic composition determination from accurate mass data by enumeration of all possible formulas within constraints defined a priori with the estimation of element coefficients from resolved isotopic structures. These estimated values allow one to narrow the search space for the composition and therefore to reduce the number of candidate formulas.

High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli

Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the ligand for NKR-P1C(B6) remains unknown. In order to initiate ligand identification, structural studies, and epitope mapping experiments, we developed a simple and efficient expression and purification protocol allowing to produce large amounts of pure soluble monomeric mouse NKR-P1C(B6). Our protein encompassed approximately half of the stalk region and the entire C-terminal globular ligand binding domain. The identity of protein that was devoid of N-terminal initiation methionine and had all three expected disulfides closed was confirmed using high resolution ion cyclotron resonance mass spectrometry. Protein produced into inclusion bodies in Escherichia coli was efficiently refolded into a unique three dimensional structure as confirmed by NMR using 1H– 15N-HSQC spectra of uniformly labeled protein. The exceptional purity of the protein should allow its crystallization and detailed structural investigations, and is a prerequisite for its use as a probe in ligand identification and antibody epitope mapping experiments.

A Gut Feeling

Medicine Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract. The exact cause is unknown, but environmental and host genetic factors are known to be important. Jansson et al. have carried out a large-scale study of the gut microbiota and detected thousands of metabolites in fecal samples from sets of identical twins that were discordant for Crohn's disease using high-resolution ion cyclotron resonance–Fourier transform mass spectrometry. They identified metabolites that could differentiate between diseased and nondiseased individuals, including some from pathways involved in tyrosine metabolism, which is consistent with genetic polymorphisms that have been associated with Crohn's disease. The data from this metabolomic study could lead to the identification of biomarkers for disease diagnosis. PLoS ONE 4 , e6386 (2009).

High-resolution ion cyclotron resonance spectroscopy of mixtures of CH 4 with N 2O and CO 2

By means of an ion cyclotron resonance (ICR) mass spectrometer operating at high mass resolution, the secondary ion signal at m/e = 29 in a N 2O/CH 4 mixture has been shown to be due to CHO + as well as N 2H + and C 2H s +. High resolution has also shown that the secondary ion CH 4N + is being produced at m/e = 30. The reactions producing CHO + in a CO 2/CH 4 mixture have also been determined at high resolution. In this mixture, a secondary ion of composition C 2H 3O + was detected at low resolution. This ion also occurs in CO/CH 4 mixtures. Precursors of this ion were determined by double resonance experiments and found to be CO 2 +, CH 4 + and CO +.