Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease.

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.

The chemodiversity of wines can reveal a metabologeography expression of cooperage oak wood.

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.

Expressing forest origins in the chemical composition of cooperage oak woods and corresponding wines by using FTICR-MS.

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.

In vitro antagonism of an actinobacterial Kitasatospora isolate against the plant pathogen Phytophthora citricola as elucidated with ultrahigh resolution mass spectrometry.

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 C(15)H(23)NO(4)+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 MS(n) 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.

Practical considerations for the analysis of ionic and neutral organic molecules with capillary electrophoresis/mass spectrometry.

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.

The hydrolysis of unsubstituted N-acylhomoserine lactones to their homoserine metabolites. Analytical approaches using ultra performance liquid chromatography.

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.

Development and application of a method for the analysis of N-acylhomoserine lactones by solid-phase extraction and ultra high pressure liquid chromatography.

A robust method based on solid-phase extraction (SPE) followed by ultra high pressure liquid chromatography (with trade name of Ultra Performance Liquid Chromatography: UPLC; Waters, Milford, MA, USA) is proposed for the determination of five derivatives of N-acylhomoserine lactones (AHLs) that play a biological role as signal molecules of several gram-negative bacteria. Different commercial SPE cartridges were tested for sample extraction, clean-up and preconcentration. Since the sample matrix was a complex growth media, careful optimization of the SPE with respect to washing procedure, elution solvent and sample solvent was necessary. No sample loss was observed when up to 100 mL spiked full media was added onto the cartridge. Applying UPLC for the determination of AHLs, the performance characteristics of the method showed good separation efficiency and high speed. In order to demonstrate the applicability of the method, supernatants with the known AHL producer Burkholderia cepacia LA3 grown in different media were investigated. Additionally, the method was successfully used for the degradation/uptake study of AHLs from a liquid matrix in which barley was grown under controlled condition.

Metabolic evidence for biogeographic isolation of the extremophilic bacterium Salinibacter ruber.

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.

CE-MS analysis of the human urinary proteome for biomarker discovery and disease diagnostics.

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. Capillary electrophoresis coupled to mass spectrometry (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 enabled 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.

At-line coupling of UPLC to chip-electrospray-FTICR-MS.

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.

Peptidomic analysis of rat urine using capillary electrophoresis coupled to mass spectrometry.

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.

Identification of bacterial N-acylhomoserine lactones (AHLs) with a combination of ultra-performance liquid chromatography (UPLC), ultra-high-resolution mass spectrometry, and in-situ biosensors.

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 beta-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.

Development of a capillary electrophoresis-mass spectrometry method for the determination of formaldehyde releasers as their hydrolysis products and amino alcohols from metal working fluids.

Metal working fluids (MWFs) are widely used as lubricants and coolants for different industrial operations. Biocides are ingredients of MWFs to control the microbial growth; derivatives of hexahydrotriazines and oxazolidines are generally used. Because of the lack of appropriate characterization, an existing capillary electrophoretic method for their quantification was improved. During the process of optimization, it became clear that hydrolysis products, derivatives of amino alcohols, severely interfere with the separation procedure. Since indirect-UV detection lacked the required selectivity, mass-selective detection was employed. NMR and MS established the absence of amino alcohols in the original educts. The aqueous solutions of the biocides stored for extended time remained amino alcohol-free, suggesting that these amino alcohols are formed from the biocides during the capillary electrophoretic separation. The observation of narrow and symmetric peaks indicated hydrolysis, and the polarity of the products implied favorable conditions for capillary electrophoretic separation. Methods were optimized for the analysis of the amino alcohols, the hydrolytic products of the formaldehyde releasers, using indirect-UV and MS detection. This method was extended to other likely solutes used as alkaline-reserve ingredients. The analytes were separated within 9 min with a high precision of migration times (the RSDs were below 1.5%). When quantifying from mobility scale, the calibration curves produced linearity with regression coefficients in the range of 0.990-0.999. The detection limit was lower than 1 mg/L in the case of MS detection. The influence of water-based MWF was also investigated, and no matrix effect on the migration of the analytes and on the peak areas was observed.

Development of a capillary electrophoretic method for the analysis of low-molecular-weight amines from metal working fluid aerosols and ambient air.

A method for the determination of low-molecular-weight amines from indoor and ambient air was developed using a concentration device followed by CE coupled with indirect spectrophotometric and mass spectrometric detection that enables a reliable, rapid-response and easy-to-operate method. In indirect detection method, the selected amines were separated from interfering metal ions and amino alcohols present in the samples with an imidazole-based buffer with ethanol and EDTA as modifier. By replacing imidazole with ammonium, the final buffer was applicable for MS detection for the analytes with m/z higher than 50. A novel monolithic polymer material based on poly(methacrylate-acrylate) copolymer was developed for sampling short-chain amines from the gaseous phase. The selected analysis conditions were applied to quantify the selected short-chain amines with detection limits for the whole procedure determined between 1 and 2 microg/filter when 40 L air was sampled with 1 L/min velocity. Improved linearity and precision were obtained when the raw, time-scaled electropherogram data were transformed into mobility-scale applied for the determination of the performance characteristics of the methods. The applicability of the process of data transformation into the mobility scale was demonstrated by studying the matrix effect of water-miscible metal working fluid (stable water-oil emulsion) and of ambient air as real samples. CE-indirect UV and CE-MS, combined with the possibility of rapid air sampling, can be useful for the estimation of short-term exposure of the selected biogenic amines.

Analysis of N-acylhomoserine lactones after alkaline hydrolysis and anion-exchange solid-phase extraction by capillary zone electrophoresis-mass spectrometry.

A quantitative, specific, and sensitive method for the determination of N-acylhomoserine lactones (HSLs - a group of bacterial semiochemicals) in the form of their hydrolysis products (N-acylhomoserines, HSs) is presented. Real samples were analyzed by capillary zone electrophoresis-mass spectrometry (CZE-MS) after alkaline lactonolysis and extraction by mixed-mode anion-exchange solid-phase extraction. The presented cleanup significantly speeds up the HSL extraction procedure, strongly reduces sample consumption, and is more selective compared to the commonly used liquid/liquid extraction. Completeness of the hydrolysis reaction was examined by nuclear magnetic resonance spectroscopy. This CZE-MS method complements recently published capillary separation techniques (nano liquid chromatography-MS, partial-filling micellar electrokinetic chromatography-MS, gas chromatography-MS) and provides a possibility to differentiate quantitatively between the homoserines (as naturally occurring degradation products) besides the intact homoserine lactones. The method was found to be quantitative down to a concentration of 0.05 microg/mL (limit of quantification), while the limit of detection was determined with 0.01 microg/mL - sufficient for the analysis of culture supernatants.

Capillary electrophoresis-mass spectrometry: 15 years of developments and applications.

Since its introduction in 1987, capillary electrophoresis-mass spectrometry (CE-MS) has developed to a well accepted multidimensional analytical approach complementary and/or competitive to classical MS-hyphenated separation techniques. The threefold combination of rapid developments of an exceptional separation technique, of selective mass detection possibilities, and of very mild ionization modes first allowed these progresses. This article shows the CE specificities that need to be well controlled/known, compared to classical and more routinely used liquid chromatography in the light of its coupling to MS. The major trends and developments over the last 15 years and most of the reviews and applications found in ISI Web of science and publisher databases are presented in a tabulated way. The reader can thus rapidly find existing CE-MS analysis techniques in his field of research and application (forensics, environment, bioanalytics, pharmaceutics, and metabolites).

A simple and robust set-up for on-column sample preconcentration--nano-liquid chromatography--electrospray ionization mass spectrometry for the analysis of N-acylhomoserine lactones.

A simple method for the simultaneous, rapid and sensitive determination of N-acylhomoserine lactone signaling molecules in bacterial isolates, without prior sample preconcentration and with minimal sample cleanup, is presented. The analysis relies on the combination of analyte preconcentration and separation on a single device: a relatively large sample volume (1-5 microL) is directly loaded onto a laboratory-made, miniaturized (75 microm i. d.) reverse phase nano-liquid chromatography column, connected on-line to a microelectrospray-ionization ion trap mass spectrometer. In a first step the analyte is adsorbed (and so concentrated) at the beginning of the column, and is eluted and selectively separated in a second step by the organic mobile phase. Sample preconcentration follows the mechanisms of solid phase extraction on a nano-scale, while separation takes place according to classical liquid chromatography separation principles. The columns can be manufactured easily, are simply connected, and used with minimal solvent amounts; this makes this method extremely robust and cost-effective. The analytical setup was found to be routinely quantitative down to a concentration of 10 ng/mL (corresponding to a total analyte amount of 10 pg or ca. 50 fmol). The limit of detection was reached at 1 ng/mL (1 pg, ca. 5 fmol). Compared to the classical AHL analysis of bacterial cultures with biosensors, where selectivity and sensitivity is often limited, this rapid analytical technique is a substantial qualitative and quantitative improvement. Two unsubstituted N-acylhomoserine lactones could be identified and quantified from a Burkholderia cepacia culture supernatant in a chloroform extract.

Analysis of N-acyl-L-homoserine lactones produced by Burkholderia cepacia with partial filling micellar electrokinetic chromatography--electrospray ionization-ion trap mass spectrometry.

A method for the analysis of N-acyl-L-homoserine lactones (AHLs) with micellar electrokinetic chromatography coupled to electrospray ionization-ion trap mass spectrometry, combining the flexibility of capillary electrophoresis with the unmatched structural information provided by mass spectrometry is presented. Different surfactants were evaluated, with sodium dodecyl sulfate (SDS) yielding the best results considering sensitivity and flexibility. We examined the interaction of AHLs with the SDS micelles at different analysis conditions and applied the optimized method to the analysis of a real bacterial sample. Two AHLs from Burkholderia cepacia colonizing the rhizosphere of traditional Indian rice cultivars could be unambiguously determined in an ethyl acetate extract with high resolution flexibility.