Metabolomics profile responses to changing environments in a common bean (Phaseolus vulgaris L.) germplasm collection.

Metabolomics is one of the most powerful -omics to assist plant breeding. Despite the recognized genetic diversity in Portuguese common bean germplasm, details on its metabolomics profiles are still missing. Aiming to promote their use and to understand the environment's effect in bean metabolomics profiles, 107 Portuguese common bean accessions, cropped under contrasting environments, were analyzed using spectrophotometric, untargeted and targeted mass spectrometry approaches. Although genotype was the most relevant factor on bean metabolomics profile, a clear genotype × environment interaction was also detected. Multivariate analysis highlighted, on the heat-stress environment, the existence of higher levels of salicylic acid, and lower levels of triterpene saponins. Three clusters were defined within each environment. White accessions presented the lowest content and the colored ones the highest levels of prenol lipids and flavonoids. Sources of interesting metabolomics profiles are now identified for bean breeding, focusing either on local or on broad adaptation.

Mining for Peaks in LC-HRMS Datasets Using Finnee - A Case Study with Exhaled Breath Condensates from Healthy, Asthmatic, and COPD Patients.

Separation techniques hyphenated to high-resolution mass spectrometry are essential in untargeted metabolomic analyses. Due to the complexity and size of the resulting data, analysts rely on computer-assisted tools to mine for features that may represent a chromatographic signal. However, this step remains problematic, and a high number of false positives are often obtained. This work reports a novel approach where each step is carefully controlled to decrease the likelihood of errors. Datasets are first corrected for baseline drift and background noise before the MS scans are converted from profile to centroid. A new alignment strategy that includes purity control is introduced, and features are quantified using the original data with scans recorded as profile, not the extracted features. All the algorithms used in this work are part of the Finnee Matlab toolbox that is freely available. The approach was validated using metabolites in exhaled breath condensates to differentiate individuals diagnosed with asthma from patients with chronic obstructive pulmonary disease. With this new pipeline, twice as many markers were found with Finnee in comparison to XCMS-online, and nearly 50% more than with MS-Dial, two of the most popular freeware for untargeted metabolomics analysis.

Background correction in separation techniques hyphenated to high-resolution mass spectrometry - Thorough correction with mass spectrometry scans recorded as profile spectra.

Separation techniques hyphenated with high-resolution mass spectrometry have been a true revolution in analytical separation techniques. Such instruments not only provide unmatched resolution, but they also allow measuring the peaks accurate masses that permit identifying monoisotopic formulae. However, data files can be large, with a major contribution from background noise and background ions. Such unnecessary contribution to the overall signal can hide important features as well as decrease the accuracy of the centroid determination, especially with minor features. Thus, noise and baseline correction can be a valuable pre-processing step. The methodology that is described here, unlike any other approach, is used to correct the original dataset with the MS scans recorded as profiles spectrum. Using urine metabolic studies as examples, we demonstrate that this thorough correction reduces the data complexity by more than 90%. Such correction not only permits an improved visualisation of secondary peaks in the chromatographic domain, but it also facilitates the complete assignment of each MS scan which is invaluable to detect possible comigration/coeluting species.

Variability of some diterpene esters in coffee beverages as influenced by brewing procedures.

Several coffee brews, including classical and commercial beverages, were analyzed for their diterpene esters content (cafestol and kahweol linoleate, oleate, palmitate and stearate) by high performance liquid chromatography with diode array detector (HPLC-DAD) combined with spectral deconvolution. Due to the coelution of cafestol and kahweol esters at 225 nm, HPLC-DAD did not give accurate quantification of cafestol esters. Accordingly, spectral deconvolution was used to deconvolve the co-migrating profiles. Total cafestol and kahweol esters content of classical coffee brews ranged from 5-232 to 2-1016 mg/L, respectively. Commercial blends contained 1-54 mg/L of total cafestol esters and 2-403 mg/L of total kahweol esters. Boiled coffee had the highest diterpene esters content, while filtered and instant brews showed the lowest concentrations. However, individual diterpene esters content was not affected by brewing procedure as in terms of kahweol esters, kahweol palmitate was the main compound in all samples, followed by kahweol linoleate, oleate and stearate. Higher amounts of cafestol palmitate and stearate were also observed compared to cafestol linoleate and cafestol oleate. The ratio of diterpene esters esterified with unsaturated fatty acids to total diterpene esters was considered as measure of their unsaturation in analyzed samples which varied from 47 to 52%. Providing new information regarding the diterpene esters content and their distribution in coffee brews will allow a better use of coffee as a functional beverage.

Algorithm for comprehensive analysis of datasets from hyphenated high resolution mass spectrometric techniques using single ion profiles and cluster analysis.

Various algorithms have been developed to improve the quantity and quality of information that can be extracted from complex datasets obtained using hyphenated mass spectrometric techniques. While different approaches are possible, the key step often consists in arranging the data into a large series of profiles known as extracted ion profiles. Those profiles, similar to mono-dimensional separation profiles, are then processed to detect potential chromatographic peaks. This allows extracting from the dataset a large number of peaks that are characteristics of the compounds that have been separated. However, with mass spectrometry (MS) detection, the response is usually a complex signal whose pattern depends on the analyte, the MS instrument and the ionization method. When converted to ionic profiles, a single separated analyte will have multiple images at different m/z range. In this manuscript we present a hierarchical agglomerative clustering algorithm to group profiles with very similar feature. Each group aims to contain all profiles that are due to the transport and monitoring of a single analyte. Clustering results are then used to generate a 2 dimensional representation, called clusters plot, which allows an in-depth analysis of the MS dataset including the visualization of poorly separated compounds even when their intensity differs by more than two orders of magnitude. The usefulness of this new approach has been validated with data from capillary electrophoresis time of flight mass spectrometry hyphenated via an electrospray ionization. Using a mixture of 17 low molecular endogenous compounds it was verified that ionic profiles belonging to each compounds were correctly clustered even with very low degree of separation (R below 0.03). The approach was also validated using a urine sample. While with the total ion profile 15 peaks could be distinguished, 70 clusters were obtained allowing a much thorough analysis. In this particular example, the total computing took less than 10 min.

Liquid chromatography with diode array detection combined with spectral deconvolution for the analysis of some diterpene esters in Arabica coffee brew.

In this manuscript, the separation of kahweol and cafestol esters from Arabica coffee brews was investigated using liquid chromatography with a diode array detector. When detected in conjunction, cafestol, and kahweol esters were eluted together, but, after optimization, the kahweol esters could be selectively detected by setting the wavelength at 290 nm to allow their quantification. Such an approach was not possible for the cafestol esters, and spectral deconvolution was used to obtain deconvoluted chromatograms. In each of those chromatograms, the four esters were baseline separated allowing for the quantification of the eight targeted compounds. Because kahweol esters could be quantified either using the chromatogram obtained by setting the wavelength at 290 nm or using the deconvoluted chromatogram, those compounds were used to compare the analytical performances. Slightly better limits of detection were obtained using the deconvoluted chromatogram. Identical concentrations were found in a real sample with both approaches. The peak areas in the deconvoluted chromatograms were repeatable (intraday repeatability of 0.8%, interday repeatability of 1.0%). This work demonstrates the accuracy of spectral deconvolution when using liquid chromatography to mathematically separate coeluting compounds using the full spectra recorded by a diode array detector.

Introducing the concept of centergram. A new tool to squeeze data from separation techniques-mass spectrometry couplings.

In separation techniques hyphenated to mass spectrometry (MS) the bulk from the separation step is continuously flowing into the mass spectrometer where the compounds, arriving at each separation time, are ionized and further separated based on their m/z ratio. An MS detector is recognized as being a universal detector, although it can also be a very selective instrument. In spite of these advantages, classical two dimensional representations from these hyphenated systems, such as those based on the base peak of electropherogram/chromatogram or on the total ion of electropherogram/chromatogram, usually hide a large number of features that if correctly assessed will show the presence of co-migrating species and/or the low abundant ones. The uses of peak picking algorithms to detect and measure as many peaks as possible from a dataset allow extracting much more information. However, a single migrating compound usually produces a multiplicity of ions, making difficult to differentiate peaks generated by the same compound from other peaks due e.g., to closely co-migrating/eluting species. In this work, a new representation is proposed and its usefulness demonstrated with experimental data from capillary electrophoresis-hyphenated to a time of flight mass spectrometer via an electrospray interface. This representation, called centergram, is obtained after using a peak picking methodology that detects electrophoretic peaks of single ions and measure their positions. The centergram is the histogram (i.e. the count of the number of observations that fall into each one of the intervals, known as bins, as determined by the user) of the measured positions. The intensity of the bars in this histogram will indicate the amount of peaks in the whole dataset whose centers are within each interval. As a compound that has been separated and has entered the MS instrument will produce multiple images at the same position along the m/z dimension, the centergram will exhibit a series of intense bars around the migration time. Those bars will allow defining a centergram peak whose area will be proportional to the number of different types of ions that have been generated in the ionization chamber, the position will be equal to the migration/retention time of the parent compounds and the width will depend on the precision in the measurement of the peak positions. The efficiency of this peak is determined to be up to thirty times higher than the equivalent peak in the classical base peak electropherogram allowing detecting easily co-migrating peaks or the presence of compounds at very low abundance. The number of peaks detected by using this new tool called centergram was increased by more than a factor of 3 compared to the standard representations.

Immobilized humic substances and immobilized aggregates of humic substances as sorbent for solid phase extraction.

In this work, humic substances (HS) immobilized, as a thin layer or as aggregates, on silica gel were tested as material for solid phase extraction. Some triazines (simazine, atrazine, therbutylazine, atrazine-desethyl-desisopropyl-2-hydroxy, ametryn and terbutryn), have been selected as test analytes due to their environmental importance and to span a large range of solubility and octanol/water partition coefficient (logP). The sorbent was obtained immobilizing a thin layer of HS via physisorption on a pre-coated silica gel with a cationic polymer (polybrene). While the sorbent could be used as it is, it was demonstrated that additional HS could be immobilized, via weak interactions, to form stable humic aggregates. However, while a higher quantity of HS could be immobilized, no significant differences were observed in the sorption parameters. This sorbent have been tested for solid phase extraction to concentrate triazines from aqueous matrixes. The sorbent demonstrated performances equivalent to commercial alternatives as a concentration factor between 50 and 200, depending on the type of triazines, was obtained. Moreover the low cost and the high flow rate of sample through the column allowed using high quantity of sorbent. The analytical procedure was tested with different matrixes including tap water, river water and estuarine water.

Dynamically formed admicelle layer to control the amplitude of cathodic electroosmotic flow.

In this manuscript, a method to precisely adjust the amplitude of a cathodic electroosmotic flow is described. The method uses a capillary pre-coated with a cationic polymer (polybrene), in presence of an anionic surfactant such as sodium dodecyl sulfate. At low concentration of surfactant, molecules will self-assemble to form an immobilized hemimicelle layer with the anionic "head" in contact with the cationic coating and the hydrophobic tail reaching into the background electrolyte. At higher concentration, surfactant molecules in solution will then interact, via hydrophobic interactions, to form an admicelle layer. It has been demonstrated that the admicelle layer can be constructed with either pure anionic surfactant (SDS), or a mixture of anionic and neutral surfactants. Admicelle coatings can be used to control the electroosmotic flow (EOF). While at low surfactant concentration the amplitude of the EOF depends on the concentration of the surfactants concentration, when this concentration reaches the critical admicelle concentration the EOF becomes near constant. The amplitude of the EOF can also be adjusted via the relative proportion of neutral and ionic surfactant in solution. Using this approach, the EOF was varied from 0.450 to 3.848 × 10(-8)m(2)V(-1)s(-1) with a precision below 0.050 × 10(-8)m(2)V(-1)s(-1) (standard deviation measured with three replicates). The coating has been tested using a mix of triazines (atrazine, simazine and terbuthylazine) and a beer sample. With the beer sample an average relative standard deviation of 1.5% for the migration time and of 2.2% for the corrected peak area was obtained.

Noise normalisation in capillary electrophoresis using a diode array detector.

When using capillary electrophoresis with a diode array detector, the wavelength at maximum absorbance is often chosen to quantify a given analyte. However, the background noise for every wavelength should be taken into account as it is by maximising the signal to noise ratio that the lowest limit of detection will be obtained. Here, we proposed an algorithm allowing to correct an electropherogram from its background absorption and to estimate the background noise. Applying it to all the electropherograms obtained in each wavelength channel allows obtaining the background noise as a function of the wavelength, which can be used to calculate the signal to noise ratio. This not only allows selecting the best wavelength to maximise the limit of detection of a given analyte, but also to generate a noise normalised base peak electropherogram (nn-BPE). It is shown that the noise normalised base peak electropherograms substantially improve the peaks visualisation. The algorithm is part of a graphic user interface that runs under MatLab environment; it does not require any programming knowledge and is freely available.

Studying the interaction between triazines and humic substances--a new approach using open tubular capillary electrochromatography.

The strength of the interaction between a pesticide and the soil organic matter is a key parameter to assess the risk of it reaching to groundwater with potentially harmful effects to human health. In this work, a new approach that allows measuring such interactions in a few minutes using a purified fraction of the soil organic matter (humic substances) is detailed. The strength of sorption is assessed via the normalised difference of elution (retention factor, k') between the chemical of interest and a neutral marker transported via electroosmotic flow through an open tubular column supporting the immobilised humic substances (open tubular capillary electrochromatography). The immobilisation was achieved by incubating a capillary, pre-coated with a monolayer of humic acid, with an acidic solution of humic substances. This induces the formation of a supramolecular structure of humic substances as it occurs in soils. This aggregate can easily be removed using alkaline solutions, and a new structure assembled using other humic substances (HS) or different incubations conditions. The whole procedure takes 2h. This approach has been tested using five triazines and three types of humic substances. The order of the strength of sorption of the triazines as expected from relevant literature and the relative standard deviation of k' was between 1 and 6%. Good repeatability was also observed after long period of wash, between re-coating and repeating of the full coating with a new capillary.

Direct photodegradation of carbamazepine followed by micellar electrokinetic chromatography and mass spectrometry.

Carbamazepine, a widely consumed psychotropic pharmaceutical, is one of the most commonly detected drugs in the environment. To better assess the environmental persistence of carbamazepine in aqueous matrices, the effect of pH and dissolved oxygen on the direct photodegradation rate of this pharmaceutical was evaluated in this study, using simulated solar irradiation. In order to follow the degradation and the emergence of photoproducts, a micellar electrokinetic chromatography based method was developed, consisting on the use of a dynamically coated capillary column. The developed methodology showed good repeatability and efficiency in the separation of carbamazepine and photoirradiation products. Also, seven photodegradation products were identified by electrospray mass spectrometry (ESI-MS), including the known carcinogenic acridine that was produced under all the pH and oxygenation levels studied and one newly identified photoproduct. This paper gives new insights into the role of dissolved oxygen on the photodegradation rate of carbamazepine. The results indicate that acidic pH, combined with the absence of dissolved oxygen in the aqueous matrix, results in very high direct photodegradation rates. At basic pH, dissolved oxygen does not interfere with the process and very low rates were observed. At environmentally relevant conditions, carbamazepine was shown to persist in the environment from 4.5 to 25 days.

Comparison between MEKC and UV spectral deconvolution to follow sorption experiment in soil.

In this study a simple and rapid procedure for monitoring sorption of atrazine onto soil samples was developed. This method is based on a multiwavelength UV spectral deconvolution (UVSD) where the UV spectrum of a sample is considered as a linear combination of absorption spectra, named reference spectra. The combination of the reference spectra allows the restitution of the shape of the UV spectrum of any unknown sample. In order to evaluate the accuracy of this method, a sorption study of atrazine to three different soil samples was monitored using both UVSD and micellar electrokinetic chromatography (MEKC) methods. The Freundlich parameters (K(F) and N) obtained for all soil samples tested were similar for both methods and UVSD has proven to be an accurate methodology, since the results did not present statistical significant differences at 95% confidence level. Moreover, the LOD obtained using UVSD presented lower values (0.066-0.12mgL(-1)) than the one obtained using MEKC (0.26mgL(-1)). The proposed UVSD method has been proven useful as a valid alternative, to the more common procedures to follow sorption experiment in soil matrix samples with no need of sample pre-treatment or column conditioning. This method is much faster and requires less sample manipulation than traditional analytical separation methods. Moreover, most modern spreadsheet-applications, such as Excel, now include statistical packages that allow performing multi-linear regression, which make this approach particularly inexpensive since the only requirement is a UV spectrometer. The regression output was made using the Microsoft Excel, a very easy and fast program, allowing anyone, with less knowledge about complex statistical programs, to perform the UV spectral.

Application of MEKC to the monitoring of atrazine sorption behaviour on soils.

Measuring interactions between soils and xenobiotics is critical to assess the threat to water resources posed by potentially harmful chemicals. Such studies are done via batch experiments where the chemical of interest is incubated with the soil for a long period of time. However, the analysis of the remaining concentration in the aqueous phase is often troublesome, due to dissolved organic matter that can interfere with the analysis. Thus, for such application a lengthy sample clean-up is often needed prior to analysis. The aim of this work was to develop an alternative approach to monitor atrazine using MEKC. A buffer of 10 mM NaH(2)PO(4) and 50 mM SDS was used to separate atrazine and soil organic matter in less than 7 min. Samples were injected without any pre-treatment leading to a significantly lower total analysis time (sample clean-up, column conditioning, separation) than HPLC. After full optimisation, good repeatability of retention time (RSD<1%) and peak areas (RSD<3%) was achieved as well as a good linearity (r(2)=0.9994) and relatively low LOD (0.26 mg/L). The method was applied to study the sorption of atrazine to a soil sample using batch equilibrium technique.

Robustness of the co-ion transfer ratio in capillary electrophoresis.

In CE, indirect detection mode often exhibits a lower precision than its direct counterpart. Although various explanations have already been advanced, in this work, we aimed to investigate if this is due, in part, to problems of robustness of the co-ion transfer ratio (TR), thus being inherent to this particular detection scheme. This was investigated using simulation software that allows an accurate control of various parameters and validated using acetic acid as a test compound. It was conclusively demonstrated that the TR could vary by more than 6% when the concentration of one of the ions in the BGE was changed by as few as 1%. The presence of a system peak seems to be particularly damaging as it has been shown that the TR of peaks whose mobilities differ by more than 0.5x10(-8) m(2) V(-1) s(-1) from one of the system peaks, still have a relatively low robustness.

Time of flight versus ion trap MS coupled to CE to analyse intact proteins.

In this work, two different CE-MS instruments, namely, CE-ESI-IT-MS and CE-ESI-TOF-MS, applied to analyse intact proteins from complex samples are investigated. The aim of this work was to compare both instruments in terms of LOD, number of proteins detected, and precision and repeatability in the determination of the protein relative molecular mass. Results show that although CE-ESI-IT-MS provides cleaner MS spectra of intact proteins, CE-ESI-TOF-MS allows the identification of a higher number of proteins from complex matrices in an easier way. Performance in terms of peak area reproducibility, LOD and precision in the determination of the molecular mass were similar for both instruments. The usefulness of the optimised CE-ESI-IT-MS and CE-ESI-TOF-MS conditions was demonstrated by studying the zein-proteins composition of three natural maize lines and their corresponding transgenic lines, showing no significant differences.

CE-MS of zein proteins from conventional and transgenic maize.

In this work, an original CE-MS method has been developed to analyze the complex zein protein fractions from maize. A thorough optimization of: (i) zein protein extraction, (ii) CE separation, and (iii) electrospray-MS (ESI-MS) detection is carried out in order to obtain highly informative CE-MS profiles of this fraction. The developed CE-MS method provides good separation of multiple zein proteins based on their electrophoretic mobilities as well as adequate characterization of these proteins based on their M(r). Zein proteins with small M(r) differences (below 100 Da) were easily separated and successfully analyzed by CE-MS. Thus, apart of the so-called 15-kDa-beta-zein and 16-kDa-gamma-zein, which are demonstrated to be formed by a heterogeneous group of proteins, numerous alpha-zeins belonging to the 19- and 22-kDa fraction were also identified for the first time in this work. The usefulness of this CE-MS method was corroborated by comparing the zein-protein fingerprints of various maize lines including transgenic and their corresponding nontransgenic isogenic lines cultivated under the same conditions.

Reproducible and efficient separation of aggregatable zein proteins by CZE using a volatile background electrolyte.

Zein proteins are a complex mixture of polypetides that belong to the alcohol-soluble storage proteins group (prolamines) in corn. These proteins constitute about 50-60% of the total endosperm protein and are classified in different groups on the basis of differences in their solubility and sequence. Among them, zein proteins are considered the majority group showing a high tendency to aggregate what makes their analysis by any analytical method very difficult. Thus, CZE of these proteins requires the use of very complex BGEs noncompatible with online ESI-MS analysis. The aim of this work was to find a new BGE for the CZE separation of zein protein fully compatible with ESI-MS while providing further light on the complex CZE separation of aggregatable proteins. Thus, it is demonstrated in this work that efficient and reproducible CZE separations of zein proteins can be achieved by using a BGE composed of water, ACN, formic acid and ammonium hydroxide. Besides, it is shown that zein analysis is significantly improved by including the effect of an ammonium gradient during their separation. It is experimentally verified that the ammonium gradient can easily be achieved in CZE by either working with a sample zone with a low concentration of ammonium and a BGE with a high concentration, or conversely, working with a sample zone with high ammonium concentration and a BGE with low concentration of ammonium, giving rise in both cases to a significant improvement in the CZE separation of these proteins. It is demonstrated that this procedure can give rise to efficiency improvements of up to 20-fold in the CZE separation of zein proteins. Under optimized conditions, 20 proteins could be separated with average efficiencies higher than 400 000 theoretical plates/m. Some possible explanations of this effect are discussed including stacking, protein-capillary wall adsorption, protein solubility and protein-salt interactions.

Simplified 2-D CE-MS mapping: analysis of proteolytic digests.

It has been demonstrated that CE-MS is a very useful hyphenated technique for proteomic studies. However, the huge amount of data stored in a single CE-MS run makes it necessary to account with procedures able to extract all the relevant information made available by CE-MS. In this work, we present a new and easy approach capable of generating a simplified 2-D map from CE-MS raw data. This new approach provides the automatic detection and characterization of the most abundant ions from the CE-MS data including their mass-to-charge (m/z) values, ion intensities and analysis times. It is demonstrated that visualization of CE-MS data in this simplified 2-D format allows: (i) an easy and simultaneous visual inspection of large datasets, (ii) an immediate perception of relevant differences in closely related samples, (iii) a rapid monitoring of data quality levels in different samples, and (iv) a fast discrimination between comigrating polypeptides and ESI-MS fragmentation ions. The strategy proposed in this work does not rely on an excellent mass accuracy for peak detection and filtering, since MS values obtained from an IT analyzer are used. Moreover, the methodology developed works directly with the CE-MS raw data, without interference by the user, giving simultaneously a simplified 2-D map and a much easier and more complete data evaluation. Besides, this procedure can easily be implemented in any CE-MS laboratory. The usefulness of this approach is validated by studying the very similar trypsin digests from bovine, rabbit and horse cytochrome c. It is demonstrated that this simplified 2-D approach allows specific markers for each species to be obtained in a fast and simple way.

Measuring the length of hydrodynamically injected plugs in capillary electrophoresis using the electrical current monitoring.

Although CE is nowadays a worldwide separation technique, it is generally recognized that one of its main limitations is its poor robustness for quantitative analysis. Although this limitation can partially be surpassed using internal standards (ISs), it is well known that to find adequate standards is a very difficult task when too complex mixtures have to be analyzed. In this work, an alternative method to improve quantitation by CE is presented using the electrical current profile monitored during any CE run. Thus, an abrupt step in the current monitoring is observed when a hydrodynamically injected plug of conductivity different from the BGE leaves the capillary under the influence of the EOF. It is demonstrated that under these conditions, the relative amplitude of this step can be used to measure experimentally the injection length. This measure can not only be used for calibration, but also to correct variations of the length injected which is demonstrated to improve significantly the quantitative accuracy and reproducibility of CE. Thus, RSD values for interday quantification (five experiments a day for 5 days) were improved from 10.5 to 4.2%. Moreover, it is also demonstrated that accuracy of quantitative determinations by CE can greatly be improved by using this procedure. The method can also be implemented in other separation techniques where the EOF is used as driving force (e.g., CEC, MEKC or chip-based separations). Advantages and limitations of this approach in comparison to the use of ISs are also discussed.