Interactions between plant growth-promoting rhizobacteria (PGPRs) and the symbiotic plant growth-promoting fungus Piriformospora indica were demonstrated by adopting several working models. Different rhizobacteria either inhibit, promote, or have no influence on stimulatory effect of P. indica in gnotobiotic barley plants. In particular, it was demonstrated that, e.g., Pseudomonas fluorescens WS5 and Burkholderia cepacia LA3 inhibited the growth and development of P. indica including complete blockage of sporulation (chlamydospores). The interaction with Ps. fluorescens turned out to be “fungistatic” as well as “fungicidal” in nature. TEM study showed the degradation of cell walls. Gas chromatography/mass spectrometry ion fragmentation pattern suggests that one of the interactive compounds is pyoverdine—a potent siderophore. On the other hand, the severe inhibition of the P. indica by the excreted metabolites of B. cepacia was described at the metabolome level applying high accurate mass spectrometer measurements. It was observed that several pathways were deactivated in the fungus, but a few of them, like ubiquinone biosynthesis, limonene, and pinene degradation, were activated since increased number of metabolites was annotated. Saponin, a biosurfactant, also inhibited the fungus but did not affect the ubiquinone biosynthesis and the limonene–pinene degradation. The study clearly demonstrated that there is intense interaction at metabolome level between rhizobacteria, P. indica, and plant components. A balance maintained due to stimulation and inhibition of the fungus by different rhizobacteria appears to be one of the major factors responsible for fungal diversity, abundance, and function in the rhizosphere. The study opens new vistas to understand delicate balance among mycorrhizospheric organisms that largely allow diverse microbes to coexist and share common resources.

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

A non-targeted, ultra-high-resolution mass spectrometric, direct analysis of oak-wood extracts from two species (Quercus robur L. and Quercus petraea Liebl.) from three French forests, and of a wine aged in barrels derived therefrom has been performed to identify families of metabolites that could discriminate both the species and the geographical origin of woods. From 12 T ultra-high-resolution Fourier transform ion cyclotron resonance mass spectra of wood extracts, hundreds of mass signals were identified as possible significant biomarkers of the two species, with phenolic and carbohydrate moieties leading the differentiation between Q. robur and Q. petraea, respectively, as corroborated by both FTMS and NMR data. For the first time, it is shown that oak woods can also be discriminated on the basis of hundreds of forest-related compounds, and particular emphasis is put on sessile oaks from the Tronçais forest, for which sugars are significantly discriminant. Despite the higher complexity and diversity of wine metabolites, forest-related compounds can also be detected in wines aged in related barrels. It is only by using these non-targeted analyses that such innovative results, which reveal specific chemodiversities of natural materials, can be obtained.

Wine chemical compositions, which result from a complex interplay between environmental factors, genetic factors, and viticultural practices, have mostly been studied using targeted analyses of selected families of metabolites. Detailed studies have particularly concerned volatile and polyphenolic compounds because of their acknowledged roles in the organoleptic and therapeutic properties. However, we show that an unprecedented chemical diversity of wine composition can be unraveled through a nontargeted approach by ultrahigh-resolution mass spectrometry, which provides an instantaneous image of complex interacting processes, not easily or possibly resolvable into their unambiguous individual contributions. In particular, the statistical analysis of a series of barrel-aged wines revealed that 10-year-old wines still express a metabologeographic signature of the forest location where oaks of the barrel in which they were aged have grown.

Introduction Recent years have witnessed the discovery of a growing number of short peptide segments that play critical biological roles. For instance, SH3 domains bind to sequences with a PxxP pattern, 14-3-3 domains to RxxSxP. Their short length (3-8 residues) and the fact that they often reside in nonglobular parts of proteins hinders their discovery both by traditional sequence comparison or experimental techniques.

Many soil microorganisms antagonistic to soil borne plant pathogens are well known for their ability to control diseases in situ. A variety of substances, like lytic enzymes, siderophores and antibiotics, produced by these organisms have the potential to protect roots against pathogens. Understanding the ecology and a functional assessment of antagonistic microbial communities in soil requires in-depth knowledge of the mechanisms involved in these interactions, a challenging task in complex systems if low-resolution methods are applied. We propose an information-rich strategy of general relevance, composed of adequate preconcentration in conjunction with ultrahigh resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS) and nuclear magnetic resonance (NMR) spectroscopy to identify any bioactive substances in complex systems. This approach is demonstrated on the specific example of substance identification considered responsible for in vitro antagonism of an actinobacterial antagonist isolated from European beech (Fagus sylvatica) rhizosphere soil against the oomycetous root rot pathogen Phytophthora citricola. The isolate belonging to the genus Kitasatospora exhibited strong antibiosis against the oomycete in vitro. The bioactive substance was observed to exhibit a molar mass of 281.1699 g/mol in positive electrospray ionization mass spectra, and the high mass accuracy of the ICR-FT/MS measurements allowed a precise assignment of a molecular formula that was found identical to the macrolide polyketide cycloheximide C15H23NO4 + H+; its identity was then unequivocally confirmed by the information-rich atomic signature of proton NMR spectroscopy. In conclusion, the combination of the near orthogonal methods (pre)fractionation, ultrahigh-resolution ICR-FT mass spectrometry (yielding molecular and MSn fragment signatures) and nuclear magnetic resonance spectroscopy (providing atomic signatures) has been found capable of identifying a biocontrol active compound of Kitasatospora active against Phytophthora citricola expediently, quickly, and accurately. This straightforward approach is of general applicability to elucidate biocontrol mechanisms in any complex system with improved efficiency.

Owing to its availability, ease of collection, and correlation with pathophysiology of diseases, urine is an attractive source for clinical proteomics. However, many proteomic studies have had only limited clinical impact, due to factors such as modest numbers of subjects, absence of disease controls, small numbers of defined biomarkers, and diversity of analytical platforms. Therefore, it is difficult to merge biomarkers from different studies into a broadly applicable human urinary proteome database. Ideally, the methodology for defining the biomarkers should combine a reasonable analysis time with high resolution, thereby enabling the profiling of adequate samples and recognition of sufficient features to yield robust diagnostic panels. CE-MS, which was used to analyze urine samples from healthy subjects and patients with various diseases, is a suitable approach for this task. The database of these datasets compiled from the urinary peptides enables the diagnosis, classification, and monitoring of a wide range of diseases. CE-MS exhibits excellent performance for biomarker discovery and allows subsequent biomarker sequencing independent of the separation platform. This approach may elucidate the pathogenesis of many diseases, and better define especially renal and urological disorders at the molecular level.

The biogeography of prokaryotes and the effect of geographical barriers as evolutionary constraints are currently subjected to great debate. Some clear- cut evidence for geographic isolation has been obtained by genetic methods but, in many cases, the markers used are too coarse to reveal subtle biogeographical trends. Contrary to eukaryotic microorganisms, phenotypic evidence for allopatric segregation in prokaryotes has never been found. Here we present, for the first time, a metabolomic approach based on ultrahigh resolution mass spectrometry to reveal phenotypic biogeographical discrimination. We demonstrate that strains of the cosmopolitan extremophilic bacterium Salinibacter ruber, isolated from different sites in the world, can be distinguished by means of characteristic metabolites, and that these differences can be correlated to their geographical isolation site distances. The approach allows distinct degrees of discrimination for isolates at different geographical scales. In all cases, the discriminative metabolite patterns were quantitative rather than qualitative, which may be an indication of geographically distinct transcriptional or posttranscriptional regulations.

This chapter presents the technique of capillary electrophoresis coupled to mass spectrometry (CE/MS). The introductory section is targeted mainly at CE/MS beginners and notes briefly the theoretical background of electrospray ionization (ESI), the most commonly used ionization mode in CE/MS. The specifics of CE/MS are described--also in comparison with more classic methods like LC/MS. Important caveats to be taken into consideration for successful CE/MS operation are noted in the interest of avoiding pitfalls. CE/MS is illustrated with three representative examples, which might serve as starting points for more in-detail experiments: (1) partial-filling micellar electrokinetic chromatography (MEKC) of neutral bacterial signaling molecules (N-acylhomoserine lactones) extracted from culture supernatants, (2) capillary zone electrophoresis (CZE) of their anionic degradation products, and finally (3) CZE separation of cationic hydroxy-s-triazines.

Owing to its availability, ease of collection and correlation with (patho-) physiology, urine is an attractive source for clinical proteomics. However, the lack of comparable datasets from large cohorts has greatly hindered development in this field. Here we report the establishment of a high resolution proteome database of naturally occurring human urinary peptides and proteins - ranging from 800-17,000 Da - from over 3,600 individual samples using capillary electrophoresis coupled to mass spectrometry, yielding an average of 1,500 peptides per sample. All processed data were deposited in an SQL database, currently containing 5,010 relevant unique urinary peptides that serve as classifiers for diagnosis and monitoring of diseases, including kidney and vascular diseases. Of these, 352 have been sequenced to date. To demonstrate the applicability of this database, two examples of disease diagnosis were provided: For renal damage diagnosis, patients with a specific renal disease were identified with high specificity and sensitivity in a blinded cohort of 131 individuals. We further show definition of biomarkers specific for immunosuppression and complications after transplantation (Kaposi’s sarcoma). Due to its high information content, this database will be a powerful tool for the validation of biomarkers for both renal and non-renal diseases.

The new sesquiterpenoid 8-deoxy-15-(3′-hydroxy-2′-methyl-propanoyl)-lactucin 3′-sulfate (1) was isolated from the methanolic extract of roots of Reichardia gaditana L. The compound was isolated by silica gel column chromatography (CC) and repeated Sephadex LH-20 CC. Structure elucidation was accomplished by high-resolution mass spectrometry and by 1D- and 2D-NMR spectroscopy. The chemosystematic significance of the new compound is discussed in the context of sesquiterpenoids from other members of the Lactuceae tribe of the Asteraceae family.

N-Acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the “quorum sensing” response of bacterial communities. Several reports have recently been published on the identification of AHLs from different species and attempts have been made to study their role in natural habitats, for example the surface of plant roots in the rhizosphere. In this article, different analytical methods, including bacterial biosensors and chromatographic techniques, are reviewed. A concept for assignment of the structures of AHLs is also presented. The retention behaviour of derivatives of AHLs containing β-keto or hydroxyl groups and/or double bonds has been evaluated in relation to the separation behaviour of AHLs with saturated and unsubstituted alkanoyl chains. Samples have also been analysed by high resolution mass spectrometry (Fourier-transform ion-cyclotron-resonance mass spectrometry, FTICR-MS), nano liquid chromatography–electrospray ionization ion trap mass spectrometry (nano-LC–MS) and by the aid of a biosensor. The results obtained from ultra performance liquid chromatography (UPLC), FTICR-MS, nano-LC–MS, and bioassays have been compared to attempt structural characterisation of AHL without chemical synthesis of analytical standards. The method was used to identify the major AHL compound produced by the rhizosphere bacterium Acidovorax sp. N35 as N-(3-hydroxydecanoyl)homoserine lactone.

The study of molecular transformation processes of dissolved organic carbon (DOC) in the environment significantly contributes to a better understanding of the global biogeochemical organic matter cycle. In an oxic karst groundwater system, in which the most powerful abiotic DOC degradative reactions, photodegradation and metal-mediated redox chemistry, are at best marginal contributors, a near complete turnover of fulvic acids (FAs) has been observed within decades (∼60 years). Depletion of oxygen for a very extensive range of aliphatic and aromatic carbon chemical environments has been confirmed as well as the formation of novel classes of compounds, suggesting a major contribution from biotic processes. From these results we infer that FAs must be perceived as a rather active participant in the global carbon cycle. Molecular-level alterations of such magnitude and rapidity on such short-time scales ought to be considered as widespread in the processing of “refractory” DOC in the environment.

Since highly sensitive on-line coupling of UPLC with FTICR-MS is technically infeasible due to their different scan rates, at-line coupling of these techniques was developed for rapid analysis. To enable cutting of one peak of the chromatogram into one fraction, several conditions and relationships were investigated, e.g. the optimum volume of the inserted delay loop, the relationship between retention time, loop outlet drop speed, individual drop volume versus mobile phase composition under constant speed, and linear solvent strength gradient elution modes. Good and reproducible results were achieved applying UPLC as an efficient separation and fast fractionation tool before the FTICR-MS measurements. A chip-based nanoelectrospray ionization system was employed which was perfectly suited to handling the small-volume fractions and was thus chosen for the at-line coupling. The method was initially applied to spiked extracts of cell-free bacterial culture supernatants in which bacterial signalling compounds, namely N-acyl homoserine lactones (AHL), were detected. Good reproducibility and high recovery was observed. Afterwards, a culture supernatant of Erwinia sp. JX3.2, a putative AHL producer, was investigated and N-hexanoyl-homoserine lactone was determined as a possible signalling molecule. More reliable assignments were achieved by use of at-line coupling of UPLC and FTICR-MS compared with off-line measurements.

We have established and validated a protocol for the peptidomic analysis of rat urine using CE coupled to MS (CE-MS). In the first experiments, the reproducibility of the CE-MS set-up and of the established preparation procedure were assessed. To establish a first rat urinary peptidome map, samples were also analyzed using CE-FT-ICR. The subsequent analysis of independent samples from two different strains (WISTAR and CD) indicated strain-specific differences, which were validated in a blinded assessment. MS/MS revealed the presence of specific fragments from well-known urinary rat peptides, such as collagens, alpha-1-antitrypsin, and serum albumin. The CE-MS-based peptidomics platform may provide novel insights into body fluids of animal models, such as rat or mice. Together with peptide identification, the technology appears to be an excellent, complimentary, and non-invasive tool to analyze toxicological or other (patho)physiological effects of pharmaceutical compounds in animal models.

Derivatives of N-acylhomoserine lactones (HSLs) with different alkanoyl side chains occur as quorum or diffusion sensing molecules in gram-negative bacteria and their quantitative chemical analysis became important as a possible way to follow regulation processes of their pathogenicity towards plants and animals. The lactone-ring of HSLs is chemically and biologically not stable: the corresponding serines can be formed in alkaline conditions and these may presumably behave inactive for the biological system. A fast and MS compatible liquid chromatographic method applying high pressure (ultra performance liquid chromatography) with diode array detection was optimized for the rapid quantitative determination of HSLs and their corresponding hydrolysis products. The technique was used to follow and model the hydrolysis reactions of HSLs as function of pH under controlled conditions. Moreover, the method could be triggered to allow a confirmation in the assignment of the potential HSLs in real samples by analysis of the real samples before and after hydrolysis. Quantitative performance characteristics and the character of the hydrolysis reaction were studied as well. The optimized method was successfully applied to a bacterial culture supernatant real sample containing HSLs.