TOPPAS: a graphical workflow editor for the analysis of high-throughput proteomics data.

Mass spectrometry coupled to high-performance liquid chromatography (HPLC-MS) is evolving more quickly than ever. A wide range of different instrument types and experimental setups are commonly used. Modern instruments acquire huge amounts of data, thus requiring tools for an efficient and automated data analysis. Most existing software for analyzing HPLC-MS data is monolithic and tailored toward a specific application. A more flexible alternative consists of pipeline-based tool kits allowing the construction of custom analysis workflows from small building blocks, e.g., the Trans Proteomics Pipeline (TPP) or The OpenMS Proteomics Pipeline (TOPP). One drawback, however, is the hurdle of setting up complex workflows using command line tools. We present TOPPAS, The OpenMS Proteomics Pipeline ASsistant, a graphical user interface (GUI) for rapid composition of HPLC-MS analysis workflows. Workflow construction reduces to simple drag-and-drop of analysis tools and adding connections in between. Integration of external tools into these workflows is possible as well. Once workflows have been developed, they can be deployed in other workflow management systems or batch processing systems in a fully automated fashion. The implementation is portable and has been tested under Windows, Mac OS X, and Linux. TOPPAS is open-source software and available free of charge at http://www.OpenMS.de/TOPPAS .

Probabilistic consensus scoring improves tandem mass spectrometry peptide identification.

Database search is a standard technique for identifying peptides from their tandem mass spectra. To increase the number of correctly identified peptides, we suggest a probabilistic framework that allows the combination of scores from different search engines into a joint consensus score. Central to the approach is a novel method to estimate scores for peptides not found by an individual search engine. This approach allows the estimation of p-values for each candidate peptide and their combination across all search engines. The consensus approach works better than any single search engine across all different instrument types considered in this study. Improvements vary strongly from platform to platform and from search engine to search engine. Compared to the industry standard MASCOT, our approach can identify up to 60% more peptides. The software for consensus predictions is implemented in C++ as part of OpenMS, a software framework for mass spectrometry. The source code is available in the current development version of OpenMS and can easily be used as a command line application or via a graphical pipeline designer TOPPAS.

BALL--biochemical algorithms library 1.3.

BACKGROUND: The Biochemical Algorithms Library (BALL) is a comprehensive rapid application development framework for structural bioinformatics. It provides an extensive C++ class library of data structures and algorithms for molecular modeling and structural bioinformatics. Using BALL as a programming toolbox does not only allow to greatly reduce application development times but also helps in ensuring stability and correctness by avoiding the error-prone reimplementation of complex algorithms and replacing them with calls into the library that has been well-tested by a large number of developers. In the ten years since its original publication, BALL has seen a substantial increase in functionality and numerous other improvements. RESULTS: Here, we discuss BALL's current functionality and highlight the key additions and improvements: support for additional file formats, molecular edit-functionality, new molecular mechanics force fields, novel energy minimization techniques, docking algorithms, and support for cheminformatics. CONCLUSIONS: BALL is available for all major operating systems, including Linux, Windows, and MacOS X. It is available free of charge under the Lesser GNU Public License (LPGL). Parts of the code are distributed under the GNU Public License (GPL). BALL is available as source code and binary packages from the project web site at http://www.ball-project.org. Recently, it has been accepted into the debian project; integration into further distributions is currently pursued.

Optimal de novo design of MRM experiments for rapid assay development in targeted proteomics.

Targeted proteomic approaches such as multiple reaction monitoring (MRM) overcome problems associated with classical shotgun mass spectrometry experiments. Developing MRM quantitation assays can be time consuming, because relevant peptide representatives of the proteins must be found and their retention time and the product ions must be determined. Given the transitions, hundreds to thousands of them can be scheduled into one experiment run. However, it is difficult to select which of the transitions should be included into a measurement. We present a novel algorithm that allows the construction of MRM assays from the sequence of the targeted proteins alone. This enables the rapid development of targeted MRM experiments without large libraries of transitions or peptide spectra. The approach relies on combinatorial optimization in combination with machine learning techniques to predict proteotypicity, retention time, and fragmentation of peptides. The resulting potential transitions are scheduled optimally by solving an integer linear program. We demonstrate that fully automated construction of MRM experiments from protein sequences alone is possible and over 80% coverage of the targeted proteins can be achieved without further optimization of the assay.

Improved particle-packed HPLC/MS microchips for proteomic analysis.

The influence of packing process parameters (packing pressure, application of ultrasound) and the stationary phase particle size (3.5 and 5 µm) on the chromatographic performance of HPLC/MS chips was systematically investigated for proteomic samples. First, reproducibility and detection limits of the separation were evaluated with a low-complexity sample of tryptic BSA peptides. The influence of adsorbent packing quality on protein identification was then tested with a typical proteomics sample of high complexity, a human plasma protein fraction (Cohn fraction IV-4). All HPLC/MS chips provided highly reproducible separations of these proteomic samples, but improved packing conditions and smaller particle sizes resulted in chromatograms with narrower peaks and correspondingly higher signal intensities. Improved separation performance increased the peak capacity, the number of identified peptides, and thus the sequence coverage in the proteomic samples, particularly for low sample amounts.

De novo peptide sequencing by tandem MS using complementary CID and electron transfer dissociation.

De novo sequencing of peptides using tandem MS is difficult due to missing fragment ions in the spectra commonly obtained after CID of peptide precursor ions. Complementing CID spectra with spectra obtained in an ion-trap mass spectrometer upon electron transfer dissociation (ETD) significantly increases the sequence coverage with diagnostic ions. In the de novo sequencing algorithm CompNovo presented here, a divide-and-conquer approach was combined with an efficient mass decomposition algorithm to exploit the complementary information contained in CID and ETD spectra. After optimizing the parameters for the algorithm on a well-defined training data set obtained for peptides from nine known proteins, the CompNovo algorithm was applied to the de novo sequencing of peptides derived from a whole protein extract of Sorangium cellulosum bacteria. To 2406 pairs of CID and ETD spectra contained in this data set, 675 fully correct sequences were assigned, which represent a success rate of 28.1%. It is shown that the CompNovo algorithm yields significantly improved sequencing accuracy as compared with published approaches using only CID spectra or combined CID and ETD spectra.

OpenMS - an open-source software framework for mass spectrometry.

BACKGROUND: Mass spectrometry is an essential analytical technique for high-throughput analysis in proteomics and metabolomics. The development of new separation techniques, precise mass analyzers and experimental protocols is a very active field of research. This leads to more complex experimental setups yielding ever increasing amounts of data. Consequently, analysis of the data is currently often the bottleneck for experimental studies. Although software tools for many data analysis tasks are available today, they are often hard to combine with each other or not flexible enough to allow for rapid prototyping of a new analysis workflow. RESULTS: We present OpenMS, a software framework for rapid application development in mass spectrometry. OpenMS has been designed to be portable, easy-to-use and robust while offering a rich functionality ranging from basic data structures to sophisticated algorithms for data analysis. This has already been demonstrated in several studies. CONCLUSION: OpenMS is available under the Lesser GNU Public License (LGPL) from the project website at http://www.openms.de.

Application of headspace and direct immersion solid-phase microextraction in the analysis of organothiophosphates related to the Chemical Weapons Convention from water and complex matrices.

The successful application of headspace (HS) and direct immersion (DI) solid phase microextraction (SPME) for the unambiguous identification and characterization of a series of toxic thiophosphate esters, such as Amiton (I), from aqueous phases and complex matrices (e.g. grass and foliage) has been demonstrated. A Thermo Scientific gas chromatograph (GC) - tandem mass spectrometer (MS/MS) system with a TriPlus RSH® autosampler and a SPME tool was used to investigate the effect of different parameters that influence the extraction efficiency: e.g. pH of the sample matrix and extraction temperature. The developed methods were employed for the detection of several Amiton derivatives (Schedule II of the CWC) that are structurally closely related to each other; some of which are new and have not been reported in literature previously. In addition, a novel DI SPME method from complex matrices for the analysis of organophosphates related to the CWC was developed. The studies clearly show that DI SPME for complex matrices is superior to HS extraction and can potentially be applied to other related compounds controlled under the CWC.

Foraging in male bumblebees (Bombus lucorum L.): maximizing energy or minimizing water load?

The O2, CO2, and H2O exchange of single flying male bumblebees (Bombus lucorum and B. terrestris) were measured simultaneously. A respiratory quotient RQ=1 was found for all activities investigated (torpor-flight). The dependence of respiratory CO2 production in flight on body-weight was measured: for a 220-mg male bumblebee it amounts to 24.5 mg CO2/h (=56.4 ml O2/g·h). The corresponding evaporative water loss amounts to 6 mg H2O/h. Males tranferred to a climatic test chamber and conditioned to artificial flower feeders started to fly, after a few days of acclimatization, in typical scent-marked flight-paths. The daily pattern of flight activity was recorded: the mean total time in flight amounts to 244 min, and the corresponding daily flight length is about 17 km. At 20°C and 50% relative humidity (RH) a daily uptake of 180 µl (≙ 220mg) of 50% sugar solution was measured, equal to the mean body weight of the male bumblebees. Since the body weight remains constant on consecutive days a 24-h energy- and water-budget could be calculated. The energy-budget is balanced; the activities observed can be fuelled with the sugar available. About 70% of the energy is used for the 4 h of flight activity. With the daily nectar volume 110 mg of water is ingested; in the oxidation of 110 mg sugar, 66 mg of metabolic water is produced and 40 mg water is dissipated by the evaporative water-loss. Thus, to have a balanced water-budget, 136 mg of water must be voided in 24 h, which equals the total body-water of the bumblebees. Nectar is a nutrient of high water content which not only provides the sugar necessary for activity but also, in most circumstances, an excess of water. The effect of this high water load in limiting daily activity is discussed and compared with the water- and osmoregulation of hummingbirds. The strategy of maximizing energy for a male bumblebee must be one of minimizing water load.

Do foraging bumblebees scent-mark food sources and does it matter?

The foraging of worker bees of Bombus terrestris visiting artificial feeders in a climatic test chamber was investigated. The behaviour of worker bees visiting rewarding and unrewarding feeders is completely different. Of all flower visits to rewarding feeders 94% are probing-visits, i.e. the bees land on the flower and probe for nectar. In contrast, only 0.3% of all visits to unrewarding feeders are probing-visits, whereas 47% are approach-visits, i.e., the bees approach the feeders without landing. Exchanging feeder discs proves that the signal used for discrimination must be associated with the plastic disc used as landing platform. Most probably it involves scent-marking of the rewarding feeders with components of high and low volatility. The mean foraging efficiency of bees in a scent-marked foraging arena is 5.7 mg sugar/min and drops to 2.8 mg sugar/min after the scent marked discs are replaced by clean ones. Three components generate this drop in foraging efficiency: (1) the between-flower flight time increases, i.e. the bees search for a longer time before landing on flowers, (2) the bees no longer discriminate between rewarding and unrewarding feeders, and (3) the bees probe empty feeders longer than necessary; obviously they expect to find nectar.

Analysis of the labial gland secretions of the male bumblebee Bombus griseocollis (Hymenoptera: Apidae).

The labial gland secretions from males of the bumblebee Bombus (Separatobombus) griseocollis De Geer, a bumblebee exhibiting perching behaviour, were analysed by gas chromatography/mass spectrometry (GC/MS) in the electron impact and positive ion chemical ionization mode. The major compound of the complex mixture of alkenols, acetates, hydrocarbons, wax type esters and steroids is tetradecyl acetate, considerable amounts of hexadecyl, geranyllinaloyl, geranylgeranyl, docosyl, tetracosenyl and hexacosenyl acetate were also found. 1,3-Tetradecanediol diacetate, detected as a minor component, has not yet been identified in male bumblebee labial gland secretions. Besides small amounts of primary alcohols (tetradecanol and hexadecanol) the tertiary alcohol geranyllinalool (3,7,11,15-tetramethyl-hexadeca-1,6,10,14-tetraene-3-ol) was also present. The primary alcohols were also present as esters of butanoic, dodecanoic, tetradecanoic, and hexadecanoic acid. Besides the usual mixture of un- and mono-unsaturated straight chain hydrocarbons, the labial gland contains the isoprenoid hydrocarbons beta-springene [(6E, 10E)-7,11,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene] and two isomers of a-springene [(3Z,6E,10E)- and (3E,6E,10E)-3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene]. The close relationship in chemical composition in male bumblebees with perching and flight pass behaviour is discussed.

Analysis of the labial gland secretions of the male bumble bee Bombus perplexus Cresson (Hymenoptera: Apidae) from North America.

The labial gland secretions from males of the bumble bee Bombus (Pyrobombus) perplexus Cresson were analysed by gas chromatography/mass spectrometry (GC/MS) in the electron impact and positive ion chemical ionization mode. The major compound of the complex mixture of alkenols, alkenals, fatty acids, hydrocarbons, wax type esters and steroids is 3,7,11,15-tetramethyl-2,6,10-hexadecatrien-1-ol (geranylcitronellol), considerable amounts of hexadecan-1-ol and Z-9-hexadecen-1-ol were also found. All alcohols were present as esters of the detected acids. In older samples both the acids and the alcohols sometimes could not be detected in the GC; therefore, the possibility to check the detected acid-alcohol pattern by interpreting the wax type ester peaks is very instructive. Moreover, the labial gland contains a rich mixture of mono- and di-unsaturated straight chain hydrocarbons. The similarity in composition of the labial glands of the North American B. (Pyrobombus) perplexus with the Eurasian species B. (Pyrobombus) hypnorum corroborates the assumption that the two species are conspecific. The likely supposition that the hydrocarbons could play an essential role in the chemical communication in bumble bees is discussed.

Displacement chromatography as first separating step in online two-dimensional liquid chromatography coupled to mass spectrometry analysis of a complex protein sample--the proteome of neutrophils.

Displacement chromatography provides some advantages over elution chromatography such as the opportunity to enrich trace amounts of molecules and to elute molecules in highest concentrations achievable with liquid chromatography. In a previous study we demonstrated that displacement chromatography is a well-suited alternative to gradient elution in an offline two-dimensional (2D-)LC-MS approach for the analysis of proteomes. In this study we present a method for applying displacement chromatography in an online 2D-LC-MS system including a cation exchange (CEX) column and a reversed phase column. We circumvented the problem of determining the sample capacity of the CEX column by repeated injection (pulses) of sample aliquots monitored by an LC-MS analysis of each flow-through fraction of the CEX column. Elution of tryptic peptides from the CEX column was achieved by repeated injection (pulses) of the displacer spermine. Pulsed displacer injections offer the advantage through physical separation of preventing post-column mixing of already separated compounds. As a proof of principle we analyzed the cytosolic proteome of human neutrophils.

OpenMS and TOPP: open source software for LC-MS data analysis.

Proteomics experiments based on state-of-the-art mass spectrometry produce vast amounts of data, which cannot be analyzed manually. Hence, software is needed which is able to analyze the data in an automated fashion. The need for robust and reusable software tools triggered the development of libraries implementing different algorithms for the various analysis steps. OpenMS is such a software library and provides a wealth of data structures and algorithms for the analysis of mass spectrometric data. For users unfamiliar with programming, TOPP ("The OpenMS Proteomics Pipeline") offers a wide range of already implemented tools sharing the same interface and designed for a specific analysis task each. TOPP thus makes the sophisticated algorithms of OpenMS accessible to nonprogrammers. The individual TOPP tools can be strung together into pipelines for analyzing mass spectrometry-based experiments starting from the raw output of the mass spectrometer. These analysis pipelines can be constructed using a graphical editor. Even complex analytical workflows can thus be analyzed with ease.

Application of displacement chromatography for the analysis of a lipid raft proteome.

Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC-MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC-MS approach.

Application of displacement chromatography for the proteome analysis of a human plasma protein fraction.

It was the aim of this study to compare the performance of displacement chromatography with gradient elution chromatography both applied as the cation-exchange separation step for a proteome analysis in a bottom-up approach using multidimensional chromatography for the separation of tryptic peptides prior to their mass spectrometric analysis. The tryptic digest of the human Cohn fraction IV-4 served as a sample. For both chromatography modes commonly used operating parameters were chosen thus ensuring optimal separation results of equal sample amounts for each mode. All resulting fractions were analyzed with an HPLC-chip-LC-MS system. The eluate of the HPLC-chip column was ionized by electrospray ionization (ESI) and analyzed with an ion-trap mass spectrometer. For guaranteeing high confidence concerning the identity of the peptides, the mass spectrometric data were processed by different bioinformatic tools applying stringent criteria. By the displacement approach the total amount of identified proteins (78) was significantly higher than in the gradient mode (58). The results showed that displacement chromatography is a well suited alternative in comparison to gradient elution separation for analysis of proteomes via the bottom-up approach applying multidimensional chromatography, especially in those cases when larger quantities of proteins are available.

Chemistry of the cephalic labial gland secretions of male Bombus morrisoni and B. rufocinctus, two North American bumblebee males with perching behavior.

The labial gland secretions from males of the North American bumblebees Bombus morrisoni Cresson and B. rufocinctus Cresson were analyzed by gas chromatography/mass spectrometry. In both species, 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl acetate was found as the major compound of a complex mixture of alkenols, acetates, hydrocarbons, and wax-type esters. In addition to a mixture of saturated and mono-unsaturated straight chain hydrocarbons, the labial gland of both species contained the isoprenoid hydrocarbons (6E, 10E)-3,7,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene [beta-springene], three isomers of 3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene [alpha-springene], and two further unidentified cyclic diterpenes. In B. morrisoni, 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol and 3,7,11,15-tetramethyl-6,10,14-hexadecatrien-1-ol were detected as characteristic alcohols, as well as small amounts of 9-hexadecenol and hexadecanol. Furthermore, a large peak of hexadecyl dodecanoate and minor amounts of 9-hexadecenyl, 9-octadecenyl, and eicosenyl 9-tetradecenoate were found as typical esters in this species. In B. rufocinctus, 9-hexadecenol, hexadecanol, and 9-octadecenol were present in considerable amounts, with their acetates and 9-tetradecenoic, tetradecanoic, hexadecanoic, and 11-octadecenoic acids. The chemical composition of cephalic labial glands in male bumblebees with perching behavior is discussed.