MyHits: improvements to an interactive resource for analyzing protein sequences.

The MyHits web site (http://myhits.isb-sib.ch) is an integrated service dedicated to the analysis of protein sequences. Since its first description in 2004, both the user interface and the back end of the server were improved. A number of tools (e.g. MAFFT, Jacop, Dotlet, Jalview, ESTScan) were added or updated to improve the usability of the service. The MySQL schema and its associated API were revamped and the database engine (HitKeeper) was separated from the web interface. This paper summarizes the current status of the server, with an emphasis on the new services.

HitKeeper, a generic software package for hit list management.

BACKGROUND: The automated annotation of biological sequences (protein, DNA) relies on the computation of hits (predicted features) on the sequences using various algorithms. Public databases of biological sequences provide a wealth of biological "knowledge", for example manually validated annotations (features) that are located on the sequences, but mining the sequence annotations and especially the predicted and curated features requires dedicated tools. Due to the heterogeneity and diversity of the biological information, it is difficult to handle redundancy, frequent updates, taxonomic information and "private" data together with computational algorithms in a common workflow. RESULTS: We present HitKeeper, a software package that controls the fully automatic handling of multiple biological databases and of hit list calculations on a large scale. The software implements an asynchronous update system that introduces updates and computes hits as soon as new data become available. A query interface enables the user to search sequences by specifying constraints, such as retrieving sequences that contain specific motifs, or a defined arrangement of motifs ("metamotifs"), or filtering based on the taxonomic classification of a sequence. CONCLUSION: The software provides a generic and modular framework to handle the redundancy and incremental updates of biological databases, and an original query language. It is published under the terms and conditions of version 2 of the GNU Public License and available at http://hitkeeper.sourceforge.net.

Characterization of lysine-guanine cross-links upon one-electron oxidation of a guanine-containing oligonucleotide in the presence of a trilysine peptide.

Formation of DNA-protein cross-links involving the initial formation of a guanine radical cation was investigated. For this purpose, riboflavin-mediated photosensitization of a TGT oligonucleotide in aerated aqueous solution in the presence of the KKK tripeptide was performed. We have shown that the nucleophilic addition of the epsilon-amino group of the central lysine residue of KKK to the C8 atom of either the guanine radical cation or its deprotonated form gives rise to the efficient formation of a Nepsilon-(guanin-8-yl)-lysine cross-link. Interestingly, the time course of formation of the above-mentioned cross-link was found to be not linear with the time of irradiation, and its formation rapidly reached a plateau. This is explained by secondary decomposition of the initially generated cross-link which could be further oxidized more efficiently than starting TGT oligonucleotide. One-electron oxidation of the initially generated cross-link was found to produce mainly two diastereomeric cross-links exhibiting a spiroimino-trilysine-dihydantoin structure as inferred from enzymatic digestion, CD, UV, NMR and mass spectrometry measurements. In addition, other minor cross-links, for which formation was favored at acidic pH, were assigned as lysine-guanine adducts in which the modified guanine base exhibits a guanidino-trilysine-iminohydantoin structure. A proposed mechanism for the formation of the different detected oligonucleotide-peptide cross-links is given. The high yield of formation of the detected cross-links strongly suggests that a DNA-protein cross-link involving a lysine residue linked to the C8 position of guanine could be generated in cellular systems if a lysine is located in the close vicinity of a guanine radical cation.

Isotope ratio monitoring of small molecules and macromolecules by liquid chromatography coupled to isotope ratio mass spectrometry.

In the field of isotope ratio mass spectrometry, the introduction of an interface allowing the connection of liquid chromatography (LC) and isotope ratio mass spectrometry (IRMS) has opened a range of new perspectives. The LC interface is based on a chemical oxidation, producing CO2 from organic molecules. While first results were obtained from the analysis of low molecular weight compounds, the application of compound-specific isotope analysis by irm-LC/MS to other molecules, in particular biomolecules, is presented here. The influence of the LC flow rate on the CO2 signal and on the observed delta13C values is demonstrated. The limits of quantification for angiotensin III and for leucine were 100 and 38 pmol, respectively, with a standard deviation of the delta13C values better than 0.4 per thousand. Also, accuracy and precision of delta13C values for elemental analyser-IRMS and flow injection analysis-IRMS (FIA-LC/MS) were compared. For compounds with molecular weights ranging from 131 to 66,390 Da, precision was better than 0.3 per thousand, and accuracy varied from 0.1 to 0.7 per thousand. In a second part of the work, a two-dimensional (2D)-LC method for the separation of 15 underivatised amino acids is demonstrated; the precision of delta13C values for several amino acids by irm-LC/MS was better than 0.3 per thousand at natural abundance. For labelled mixtures, the coefficient of variation was between 1% at 0.07 atom % excess (APE) for threonine and alanine, and around 10% at 0.03 APE for valine and phenylalanine. The application of irm-LC/MS to the determination of the isotopic enrichment of 13C-threonine in an extract of rat colon mucosa demonstrated a precision of 0.5 per thousand, or 0.001 atom %.

Glucuronidation of the green tea catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes.

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent (12.2 +/- 0.2 and 7.5 +/- 0.2%, respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.

In-depth mechanistic study on the formation of acrylamide and other vinylogous compounds by the maillard reaction.

The formation of acrylamide was studied in low-moisture Maillard model systems (180 degrees C, 5 min) based on asparagine, reducing sugars, Maillard intermediates, and sugar degradation products. We show evidence that certain glycoconjugates play a major role in acrylamide formation. The N-glycosyl of asparagine generated about 2.4 mmol/mol acrylamide, compared to 0.1-0.2 mmol/mol obtained with alpha-dicarbonyls and the Amadori compound of asparagine. 3-Hydroxypropanamide, the Strecker alcohol of asparagine, generated only low amounts of acrylamide ( approximately 0.23 mmol/mol), while hydroxyacetone increased the acrylamide yields to more than 4 mmol/mol, indicating that alpha-hydroxy carbonyls are much more efficient than alpha-dicarbonyls in converting asparagine into acrylamide. The experimental results are consistent with the reaction mechanism based on (i) a Strecker type degradation of the Schiff base leading to azomethine ylides, followed by (ii) a beta-elimination reaction of the decarboxylated Amadori compound to afford acrylamide. The beta-position on both sides of the nitrogen atom is crucial. Rearrangement of the azomethine ylide to the decarboxylated Amadori compound is the key step, which is favored if the carbonyl moiety contains a hydroxyl group in beta-position to the nitrogen atom. The beta-elimination step in the amino acid moiety was demonstrated by reacting under low moisture conditions decarboxylated model Amadori compounds obtained by synthesis. The corresponding vinylogous compounds were only generated if a beta-proton was available, for example, styrene from the decarboxylated Amadori compound of phenylalanine. Therefore, it is suggested that this thermal pathway may be common to other amino acids, resulting under certain conditions in their respective vinylogous reaction products.

Detection of Amadori compounds by capillary electrophoresis coupled to tandem mass spectrometry.

Capillary electrophoresis coupled to mass spectrometry (CE-MS) is reported for the first time as an alternative and powerful analytical method for the characterization and monitoring of N-substituted 1-amino-1-deoxyketoses (Amadori compounds). It allows rapid separation and identification of Amadori compounds, while benefiting from the well-known advantages of MS, such as specificity and sensitivity. Amadori compounds of several amino acids, such as glycine, valine, isoleucine, methionine, proline, and phenylalanine, as well as a cysteine-derived compound, were separated and/or discriminated using CE-MS/MS under standard conditions. The technique may also be useful to study the stability and degradation kinetics of other labile charged Maillard intermediates that play an important role in food and medical science.

A new technique (COMSPARI) to facilitate the identification of minor compounds in complex mixtures by GC/MS and LC/MS: tools for the visualization of matched datasets.

In the rapidly growing field of metabolomics, it is common to analyze complex biological samples by chromatography coupled to mass spectrometry. While several techniques are available for the detection of significant peaks in individual samples, it is still difficult to determine small differences between similar samples. Using conventional software, visual inspections of individual chromatograms or individual mass spectra are often of little use because the differences in the composition of small molecules are too small to be recognizable. Thus, we developed a new approach to visualizing mass spectral datasets using a tool that allows one to easily detect these small differences between mass spectra and chromatograms derived from matched samples. Using these tools on extracts from wild-type and methyltransferase knockout strains of the yeast Saccharomyces cerevisiae, we were able to readily identify those mass spectra in our data sets that were different between the wild-type and the knockout extracts and to identify the molecules involved. The software was also successfully applied to a set of LC/MS data from peptide digests that were performed with identical substrates but different enzymes. We have named this visualization tool COMSPARI (COMparision of SPectrAl Retention Information) and are making the software publicly available via Internet at.

Quantitative analysis of triacylglycerol regioisomers in fats and oils using reversed-phase high-performance liquid chromatography and atmospheric pressure chemical ionization mass spectrometry.

Positional distribution of fatty acyl chains of triacylglycerols (TGs) in vegetable oils and fats (palm oil, cocoa butter) and animal fats (beef, pork and chicken fats) was examined by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to atmospheric pressure chemical ionization using a quadrupole mass spectrometer. Quantification of regioisomers was achieved for TGs containing two different fatty acyl chains (palmitic (P), stearic (S), oleic (O), and/or linoleic (L)). For seven pairs of 'AAB/ABA'-type TGs, namely PPS/PSP, PPO/POP, SSO/SOS, POO/OPO, SOO/OSO, PPL/PLP and LLS/LSL, calibration curves were established on the basis of the difference in relative abundances of the fragment ions produced by preferred losses of the fatty acid from the 1/3-position compared to the 2-position. In practice the positional isomers AAB and ABA yield mass spectra showing a significant difference in relative abundance ratios of the ions AA(+) to AB(+). Statistical analysis of the validation data obtained from analysis of TG standards and spiked oils showed that, under repeatability conditions, least-squares regression can be used to establish calibration curves for all pairs. The regression models show linear behavior that allow the determination of the proportion of each regioisomer in an AAB/ABA pair, within a working range from 10 to 1000 microg/mL and a 95% confidence interval of +/-3% for three replicates.

Acrylamide from Maillard reaction products.

The discovery of the adventitious formation of the potential cancer-causing agent acrylamide in a variety of foods during cooking has raised much concern, but the chemical mechanism(s) governing its production are unclear. Here we show that acrylamide can be released by the thermal treatment of certain amino acids (asparagine, for example), particularly in combination with reducing sugars, and of early Maillard reaction products (N-glycosides). Our findings indicate that the Maillard-driven generation of flavour and colour in thermally processed foods can -- under particular conditions -- be linked to the formation of acrylamide.

Alkylpyridiniums. 1. Formation in model systems via thermal degradation of trigonelline.

Trigonelline is a well-known precursor of flavor/aroma compounds in coffee and undergoes significant degradation during roasting. This study investigates the major nonvolatile products that are procured after trigonelline has been subjected to mild pyrolysis conditions (220-250 degrees C) under atmospheric pressure. Various salt forms of trigonelline were also prepared and the thermally produced nonvolatiles analyzed by thin layer chromatography, liquid chromatography-electrospray ionization tandem mass spectrometry, and (1)H and (13)C nuclear magnetic resonance. Results revealed the decarboxylated derivative 1-methylpyridinium as a major product of certain salts, the formation of which is positively correlated to temperature from 220 to 245 degrees C. Moreover, trigonelline hydrochloride afforded far greater amounts of 1-methylpyridinium compared to the monohydrate over the temperature range studied. Investigations into other potential quaternary amine products of trigonelline also indicate nucleophilic substitution reactions that lead to dialkylpyridiniums, albeit at concentration levels approximately 100-fold lower than those recorded for 1-methylpyridinium.