High-throughput Environment Research Articles

Identification of Protein Modifications Using MS/MS de Novo Sequencing and the OpenSea Alignment Algorithm

Algorithms that can robustly identify post-translational protein modifications from mass spectrometry data are needed for data-mining and furthering biological interpretations. In this study, we determined that a mass-based alignment algorithm (OpenSea) for de novo sequencing results could identify post-translationally modified peptides in a high-throughput environment. A complex digest of proteins from human cataractous lens, a tissue containing a high abundance of modified proteins, was analyzed using two-dimensional liquid chromatography, and data was collected on both high and low mass accuracy instruments. The data were analyzed using automated de novo sequencing followed by OpenSea mass-based sequence alignment. A total of 80 modifications were detected, 36 of which were previously unreported in the lens. This demonstrates the potential to identify large numbers of known and previously unknown protein modifications in a given tissue using automated data processing algorithms such as OpenSea.

Electrophoresis microchips for DNA analysis

Microfabricated bioanalytical devices are very efficient platforms for simultaneous analysis of biologically important molecules; they offer great potential for sensitive and large-scale DNA analysis. Indeed, electrophoresis-based sizing of nucleic acids is a leading application in microscale analysis. Adopting the concepts of conventional capillary electrophoresis, and various sample-preparation methods to microchip format, in conjunction with integrating the different analysis steps into a single monolithic system has opened up new levels in performance, functionality and throughput. Microfabricated bioanalytical devices, frequently referred to as “lab-on-a-chip”, typically comprise microchannel networks, miniaturized polymerase chain reaction chambers and/or restriction digestion reactors, microseparation units and possible combinations of the above to accommodate DNA analysis. Some of the main advantages of miniaturization include improved performance, separation speed and throughput, reduced costs and reagent consumption, as well as integrated and parallel analysis for high-throughput environments. This review summarizes the recent advances in the field of microfabricated separation devices for genomics applications; it also addresses system integration and micropreparative functionalities.

Enhanced method performance due to a shorter chromatographic run-time in a liquid chromatography–tandem mass spectrometry assay for paclitaxel

Liquid chromatography–tandem mass spectrometry (LC–MS–MS) is often performed in a high-throughput environment. Unfortunately, with atmospheric pressure ionization (API) techniques, shorter run-times or reduced sample clean-up often result in ion or matrix suppression, which can lead to erroneous results. The present work on the analysis of paclitaxel compares ion suppression, sensitivity and linearity of a high-throughput LC–MS–MS method (0.8 min run-time, method B) to a method with increased separation (2.0 min run-time, method A). An atmospheric pressure chemical ionization (APCI) interface was used for both methods. The high-sample-throughput method uses an increased amount of organic solvent in the mobile phase (isocratic, 85% versus 70% of methanol) and a higher flow-rate (600 μl/min versus 400 μl/min). As a result, internal standard (docetaxel) and target analyte (paclitaxel) co-elute, close to, although separated from the solvent front. Ion suppression of both methods was evaluated by comparing pure standard to plasma and plasma containing a vehicle. Sensitivity and linearity were compared by injecting matrix matched calibration samples with both methods. Ion suppression by the vehicle Cremophor EL led to poor data quality for the standard method (A), while for the short method (B), ion suppression was compensated for by the co-eluting internal standard. The short method showed similar linearity but increased sensitivity by at least a factor five. This work provides a strategy to compensate for ion suppression without the use of labeled internal standards. In addition, a better sensitivity and a shorter run-time are noted.

Enhanced method performance due to a shorter chromatographic run-time in a liquid chromatography?tandem mass spectrometry assay for paclitaxel

Liquid chromatography–tandem mass spectrometry (LC–MS–MS) is often performed in a high-throughput environment. Unfortunately, with atmospheric pressure ionization (API) techniques, shorter run-times or reduced sample clean-up often result in ion or matrix suppression, which can lead to erroneous results. The present work on the analysis of paclitaxel compares ion suppression, sensitivity and linearity of a high-throughput LC–MS–MS method (0.8 min run-time, method B) to a method with increased separation (2.0 min run-time, method A). An atmospheric pressure chemical ionization (APCI) interface was used for both methods. The high-sample-throughput method uses an increased amount of organic solvent in the mobile phase (isocratic, 85% versus 70% of methanol) and a higher flow-rate (600 μl/min versus 400 μl/min). As a result, internal standard (docetaxel) and target analyte (paclitaxel) co-elute, close to, although separated from the solvent front. Ion suppression of both methods was evaluated by comparing pure standard to plasma and plasma containing a vehicle. Sensitivity and linearity were compared by injecting matrix matched calibration samples with both methods. Ion suppression by the vehicle Cremophor EL led to poor data quality for the standard method (A), while for the short method (B), ion suppression was compensated for by the co-eluting internal standard. The short method showed similar linearity but increased sensitivity by at least a factor five. This work provides a strategy to compensate for ion suppression without the use of labeled internal standards. In addition, a better sensitivity and a shorter run-time are noted.

Robust STR multiplexes for challenging casework samples

The presence of amplification inhibitors and degraded DNA in many World Trade Center (WTC) victim bone fragment samples motivated us to create STR multiplex sets displaying more robust amplification with challenging casework samples. We developed multiplex sets that co-amplify smaller versions of the larger amplicons present in commercial sets. They generate well-balanced profiles with at least five-fold the typical signal using 0.25 ng of human DNA template and generated three times the genetic information derived from commercial kits using the more challenging WTC samples. Sensitivity of the systems approaches detection of individual DNA fragment templates, requiring implementation of appropriate interpretation guidelines for this low copy number, high throughput environment.

High-Throughput Identification of Proteins and Unanticipated Sequence Modifications Using a Mass-Based Alignment Algorithm for MS/MS de Novo Sequencing Results

With the increasing availability of de novo sequencing algorithms for interpreting high-mass accuracy tandem mass spectrometry (MS/MS) data, there is a growing need for programs that accurately identify proteins from de novo sequencing results. De novo sequences derived from tandem mass spectra of peptides often contain ambiguous regions where the exact amino acid order cannot be determined. One problem this poses for sequence alignment algorithms is the difficulty in distinguishing discrepancies due to de novo sequencing errors from actual genomic sequence variation and posttranslational modifications. We present a novel, mass-based approach to sequence alignment, implemented as a program called OpenSea, to resolve these problems. In this approach, de novo and database sequences are interpreted as masses of residues, and the masses, rather than the amino acid codes, are compared. To provide further flexibility, the masses can be aligned in groups, which can resolve many de novo sequencing errors. The performance of OpenSea was tested with three types of data: a mixture of known proteins, a mixture of unknown proteins that commonly contain sequence variations, and a mixture of posttranslationally modified known proteins. In all three cases, we demonstrate that OpenSea can identify more peptides and proteins than commonly used database-searching programs (SEQUEST and ProteinLynx) while accurately locating sequence variation sites and unanticipated posttranslational modifications in a high-throughput environment.

Extractor for ESI quadrupole TOF tandem MS data enabled for high throughput batch processing

BackgroundMass spectrometry based proteomics result in huge amounts of data that has to be processed in real time in order to efficiently feed identification algorithms and to easily integrate in automated environments. We present wiff2dta, a tool created to convert MS/MS data obtained using Applied Biosystem's QStar and QTrap 2000 and 4000 series.ResultsComparing the performance of wiff2dta with the standard tools, we find wiff2dta being the fastest solution for extracting spectrum data from ABIs raw file format. wiff2dta is at least 10% faster than the standard tools. It is also capable of batch processing and can be easily integrated in high throughput environments. The program is freely available via , and is also available from Applied Biosystems.Conclusionswiff2dta offers the possibility to run as stand-alone application or within a batch process as command-line tool integrated in automation and high-throughput environments. It is more efficient than the state-of-the-art tools provided.

Optimal on-line sampling of parallel reactions: general concept and a specific spectroscopic example

The ‘gap’ between the discovery and the optimisation phases in high-throughput systems is described and the problems that stem from the centralised nature of parallel reactor set-ups are outlined. It is proposed that on-line analysis methods (e.g. fast spectroscopic analysis) can be adapted to perform kinetic studies in high-throughput environments. Strategies for performing time-resolved analysis are examined using a (pseudo)first-order reaction that is monitored by on-line FT-NIR as an example. Stochastic modelling is used to compare the efficiency of the various strategies in the determination of accurate first-order kinetic constants. The effects of the sampling time, the ‘sample window’ size, and the number of samples on the error in k obs are studied. A new sampling strategy is introduced that is based on equidistant sampling along the concentration axis. This strategy is adapted to realistic conditions using a simple iterative algorithm. The application of this iterative approach to automatic intelligent on-line analysis of chemical reaction kinetics is discussed.

Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards.

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.

High‐throughput logP measurement using parallel liquid chromatography/ultraviolet/mass spectrometry and sample‐pooling

A novel approach to high-throughput logP measurement based on liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS) is proposed. The logP value is determined by correlation with the logk value, where k is the capacity factor k = (t(r)-t(0))/t(0), with the logP value using a defined set of standards. Since the analyte retention time (t(r)) is determined from the appropriate extracted ion chromatogram (EIC), there are no interferences from impurities and this allows the pooling of multiple compounds into one injection. To ensure the accuracy and instrument robustness in a routine high-throughput environment, a simple and MS-friendly mobile phase consisting of 20 mM ammonium carbonate (pH 8.0) for basic compounds or 20 mM ammonium formate (pH 1.0) for acidic compounds, both in combination with methanol at a ratio of 45:55, is used. This approach has been successfully used on single as well as parallel multi-channel LC/UV/MS systems to screen small to large sets of lead compounds and their analogs. A high-throughput capability to analyze over 1000 compounds per day has been achieved.

Planar waveguides for ultra-high sensitivity of the analysis of nucleic acids

In the first part of this paper, the need for analytical techniques capable of highly parallel and sensitive nucleic acid analysis, with the capability of achieving very low limits of detection (LODs) and of resolving small differences in concentration, is described. Whereas the requirement for performing simultaneously multi-analyte detection is solved by the approach of nucleic acid microarrays, requirements on sensitivity can often not be satisfied by classical detection technologies. Inherent limitations of conventional fluorescence excitation and detection schemes are identified, and the implementation of planar waveguides as analytical platforms for nucleic acid microarrays, with fluorescence excitation in the evanescent field associated with the guided excitation light, is proposed. The relevant parameters for an optimization of sensitivity are discussed. In the second part of this paper, the specific formats of our planar waveguide platforms, which are compatible with established industrial standard formats allowing for integration into industrial high throughput environments, are presented, as well as the dedicated optical system for fluorescence excitation and detection that we developed. In a direct comparison with a state-of-the-art scanner, it is demonstrated that the implementation of genomic microarrays on planar waveguide platforms allows for unprecedented, direct detection of low-abundant genes in limited amounts of sample. Otherwise, when using conventional fluorescence excitation and detection configurations, the detection of such low amounts of nucleic acids requires massive sample preparation and signal or target amplification steps.

Surviving and thriving in the high throughput environment

Surviving and thriving in the high throughput environment

Design of high-throughput methods of protein production for structural biology

I greatly appreciate interacting with Peter Schultz, Scott Lesley and Marc Nasoff at the Genomics Institute for the Novartis Research Foundation and Mark Knuth and Ron Swanson at Syrrx, Inc., where much of this work is currently being pursued. I also thank Jim Graziano, Chris Lee, Mike Hanson and Marianne Patch for helpful suggestions on this manuscript.

A 32-Bit, 200-MHz GaAs RISC for High-Throughput Signal Processing Environments

GaAs technology has matured sufficiently to allow fabrication of an entire RISC on one chip. GaAs also supports 200-MHz clock rates and 100-MIPS instruction rates.