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Analytica Chimica Acta (v.564, #1)

Outside front cover - Editorial Board (pp. co1).

Contents (pp. vii-viii).

Calendar of forthcoming meetings (pp. n1).

Outside front cover - Editorial Board (pp. co1).

Contents (pp. vii-viii).

Calendar of forthcoming meetings (pp. n1).

High-resolution protein hydration NMR experiments: Probing how protein surfaces interact with water and other non-covalent ligands by Hao Huang; Giuseppe Melacini / (pp. 1-9).

High-resolution solution NMR experiments are extremely useful to characterize the location and the dynamics of hydrating water molecules at atomic resolution. However, these methods are severely limited by undesired incoherent transfer pathways such as those arising from exchange-relayed intra-molecular cross-relaxation. Here, we review several complementary exchange network editing methods that can be used in conjunction with other types of NMR hydration experiments such as magnetic relaxation dispersion and¹ J_NC′ measurements to circumvent these limitations. We also review several recent contributions illustrating how the original solution hydration NMR pulse sequence architecture has inspired new approaches to map other types of non-covalent interactions going well beyond the initial scope of hydration. Specifically, we will show how hydration NMR methods have evolved and have been adapted to binding site mapping, ligand screening, protein–peptide and peptide–lipid interaction profiling.

Keywords: Abbreviations; CT; constant time; CTJ; CT quadrature-free; J; -resolved dimension; DHPC; dihexanoyl phosphatylcholine; DMF; N; ,; N; -dimethylformamide; DMPC; dimyristoil phosphatylcholine; DMSO; dymethyl-sulfoxide; HIV; human immunodeficiency virus; IR; infra red; MD; molecular dynamics; HMQC; hetero-nuclear multiple quantum coherence spectrum; HSQC; hetero-nuclear single quantum coherence spectrum; MRD; magnetic relaxation dispersion; MS; mass spectrometry; NMR; nuclear magnetic resonance; NOE; nuclear overhauser effect; NOESY; 2D-NOE spectroscopy; PDB; protein data bank; PFG; pulsed field gradient; QF; quadrature-free; ROE; rotating frame overhauser effect; ROESY; 2D-ROE spectroscopy; TROSY; transverse relaxation optimized spectroscopy; XH; proton exchangeable with waterProtein; Water; Hydration; NMR; NOE; ROE; Surface; Screening

High-resolution protein hydration NMR experiments: Probing how protein surfaces interact with water and other non-covalent ligands by Hao Huang; Giuseppe Melacini / (pp. 1-9).

Analysis of protein interaction networks using mass spectrometry compatible techniques by Martin Ethier; Jean-Philippe Lambert; Julian Vasilescu; Daniel Figeys (pp. 10-18).

The ability to map protein–protein interactions has grown tremendously over the last few years, making it possible to envision the mapping of whole or targeted protein interaction networks and to elucidate their temporal dynamics. The use of mass spectrometry for the study of protein complexes has proven to be an invaluable tool due to its ability to unambiguously identify proteins from a variety of biological samples. Furthermore, when affinity purification is combined with mass spectrometry analysis, the identification of multimeric protein complexes is greatly facilitated. Here, we review recent developments for the analysis of protein interaction networks by mass spectrometry and discuss the integration of different bioinformatic tools for predicting, validating, and managing interaction datasets.

Keywords: Proteomics; Mass spectrometry; Protein interactions; Immunoprecipitation

Analysis of protein interaction networks using mass spectrometry compatible techniques by Martin Ethier; Jean-Philippe Lambert; Julian Vasilescu; Daniel Figeys (pp. 10-18).

Keywords: Proteomics; Mass spectrometry; Protein interactions; Immunoprecipitation

Proteomics in cancer research: Methods and application of array-based protein profiling technologies by Ann Hoeben; Bart Landuyt; Gehan Botrus; Gert De Boeck; Gunther Guetens; Martin Highly; Allan T. van Oosterom; Ernst A. de Bruijn (pp. 19-33).

With the human genome sequence now determined, the field of molecular medicine is moving beyond genomics to proteomics, the large-scale analysis of proteins.It is now possible to examine the expression of more than 1000 proteins using mass spectrometry technology coupled with various separation methods.Microarray technology is a new and efficient approach, for extracting relevant biomedical data and has a wide range of applications. It provides a versatile tool to study protein–protein, protein–nucleic acid, protein–lipid, enzyme–substrate and protein–drug interactions.This review paper will explore the key themes in proteomics and their application in clinical cancer research.

Keywords: Proteomics; Protein arrays; Tissue microarray; Cell array; Cancer research

Duplexed sandwich immunoassays on a fiber-optic microarray by David M. Rissin; David R. Walt (pp. 34-39).

In this paper, we describe a duplexed imaging optical fiber array-based immunoassay for immunoglobulin A (IgA) and lactoferrin. To fabricate the individually addressable array, microspheres were functionalized with highly specific monoclonal antibodies. The microspheres were loaded in microwells etched into the distal face of an imaging optical fiber bundle. Two microsphere-based sandwich immunoassays were developed to simultaneously detect IgA and lactoferrin, two innate immune system proteins found in human saliva. Individual microspheres could be interrogated for the simultaneous measurement of both proteins. The working concentration range for IgA detection was between 700pM and 100nM, while the working concentration range for lactoferrin was between 385pM and 10nM. The cross-reactivity between detection antibodies and their non-specific targets was relatively low in comparison to the signal generated by the specific binding with their targets. These results suggest that the degree of multiplexing on this fiber-optic array platform can be increased beyond a duplex.

Keywords: Microarray; Sandwich immunoassay; High-throughput; Protein array

Duplexed sandwich immunoassays on a fiber-optic microarray by David M. Rissin; David R. Walt (pp. 34-39).

Keywords: Microarray; Sandwich immunoassay; High-throughput; Protein array

Active bead-linked immunoassay on protein microarrays by Victor N. Morozov; Tamara Ya. Morozova (pp. 40-52).

Protein microarrays are becoming a powerful tool in proteome, biochemical, and clinical studies. In addition to the quality of arrayed immobilized probe molecules, sensitivity of the microarray-based assay is highly dependent on the detection technique. Here we suggest four simple techniques for rapid detection of analytes bound to protein microarrays. The techniques employ functionalized magnetic and non-magnetic beads moved to, from, or along the array surface by external forces. In contrast to other labeling techniques actively controlled physical labels: (i) make detection extremely fast to allow microarray reading in seconds; (ii) provide a low background due to active removal of weakly bound beads; and (iii) provide a highly sensitive detection, since one antigen–antibody bond is capable of holding bead immobilized on the array surface. In combination with the electrophoretically assisted active immunoassay we described recently such active reading allows to reduce total indirect immunoassay time to 7–10min while having sensitivity in the femtomolar concentration range. High speed, sensitivity, and specificity make active bead-linked detection an ideal choice in rapid high-throughput screening and in emergency diagnostics.

Keywords: Abbreviations; A; protein A; AAD; adipic acid dihydrazide; a-mIgG; anti-mouse-IgG; anti-rIgG-AP; antibody against rabbit IgG conjugated with alkaline phosphatase; anti-HEWL-IgG; antibody against hen egg white lysozyme; anti-R; N; -IgG; antibody against pancreatic ribonuclease; anti-HK-IgG; antibody against yeast hexokinase; anti-Ova-IgG; antibody against ovalbumin; AFM; atomic force microscopy; AP; alkaline phosphatase; B; biotin; b-a-mIgG; biotinylated anti-mouse-IgG; BCIP/NBT; 5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazoliuum; EDC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; ELISA; enzyme-linked immunosorbent assay; EP; electrophoresis; ESD; electrospray deposition; FITC; fluorescein isothiocyanate; FITC–BSA; cross-linked bovine serum albumin modified with FITC; G; protein G; GG-T; N; -Glycylglycine buffer with Tween-20; HEWL; hen egg white lysozyme; hIgG; human IgG; HK; yeast hexokinase; LOD; limit of detection; MES; 2-Morpholinoethanesulfonic acid; mIgG; mouse IgG; NHS; N; -hydroxysuccinimide; PBS; phosphate buffered saline; rIgG; rabbit IgG; RN; pancreatic RNAse; SA; streptavidin; SA–AP; streptavidin conjugated with alkaline phosphatase; TBS; Tris buffered saline; Ova; ovalbuminMagnetic beads; Protein microarray; Electrospray deposition; Active assay

Active bead-linked immunoassay on protein microarrays by Victor N. Morozov; Tamara Ya. Morozova (pp. 40-52).

Microarray techniques for more rapid protein quantification: Use of single spot multiplex analysis and a vibration reaction unit by Yukie Sasakura; Katsuhiro Kanda; Shinichi Fukuzono (pp. 53-58).

Protein microarray technology is a powerful, popular tool for the high-throughput analysis of protein interactions. One important use for protein microarray technology is protein quantification by immunoassay, which was originally based on enzyme linked immunosorbent assay (ELISA) methods. Recently, new research and diagnostic applications have created a need for a rapid and easily applied high-throughput protein quantification method. Here, we introduce several novel techniques that address these needs. Our improved protein microarray-based sandwich immunoassay techniques allow researchers to: (1) control the size and shape of protein spots on the microarray using a perforated seal; (2) analyze two proteins within a single spot, thus increasing the number of tests run on a single microarray without increasing the number of protein spots; (3) improve the efficiency and speed of the Ag–Ab interaction through vibratory reagent convection, which increased the signal intensity by more than two-fold and decreased the reaction time from 30 to 10min. These new techniques will facilitate rapid immunoassays for diagnostic purposes and other research areas utilizing protein microarray analysis, such as investigations of ligand–receptor or protein–small molecule interactions.

Keywords: Microarray; Immunoassay; Multiplex; PDMS

Microarray techniques for more rapid protein quantification: Use of single spot multiplex analysis and a vibration reaction unit by Yukie Sasakura; Katsuhiro Kanda; Shinichi Fukuzono (pp. 53-58).

Keywords: Microarray; Immunoassay; Multiplex; PDMS

Templated xerogels as platforms for biomolecule-less biomolecule sensors by Zunyu Tao; Elizabeth C. Tehan; Rachel M. Bukowski; Ying Tang; Ellen L. Shughart; William G. Holthoff; Alexander N. Cartwright; Albert H. Titus; Frank V. Bright (pp. 59-65).

We report on a new sensor strategy that we have termed protein imprinted xerogels with integrated emission sites (PIXIES). The PIXIES platform is completely self-contained, and it achieves analyte recognition without a biorecognition element (e.g., antibody). The PIXIES relies upon sol–gel-derived xerogels, molecular imprinting, and the selective installation of a luminescent reporter molecule directly within the molecularly imprint site. In operation the templated xerogel selectively recognizes the target analyte, the analyte binds to the template site, and binding causes a change in the physicochemical properties within the template site that are sensed and reported by the luminescent probe molecule. We report the PIXIES analytical figures of merit for and compare these results to a standard ELISA. For human interleukin-1 the PIXIES-based sensor elements exhibited the following analytical figures of merit: (i) ∼2pg/mL detection limits; (ii) <2min response times; (iii) >85 selectivity; (iv) <6% R.S.D. long term drift over 16 weeks of ambient storage; (v) >95% reversibility after more than 25 cycles; and (vi) >85% recoveries on spiked samples.

Keywords: Sensors; Xerogels; Arrays; Protein detection; Molecular imprinting; Robotic screening

Increasing robustness and sensitivity of protein microarrays through microagitation and automation by M. Hartmann; A. Toegl; R. Kirchner; M.F. Templin; T.O. Joos (pp. 66-73).

Assay systems that employ protein microarrays for the analysis of complex samples are powerful tools to generate a high amount of data from a limiting amount of sample. Due to miniaturization, these systems are susceptible to fluctuations during signal generation and the use of uniform conditions for sample incubation and during the assay procedure is required to get reproducible results. Diffusion limits may prevent constant conditions on all parts of the array and can lead to the decease of the sensitivity of the array. Therefore, we set-up an automated assay system integrating a novel microagitation device using surface acoustic wave (SAW) technology. Multiplexed assays for the detection of autoantibodies from human serum and sandwich immunoassay for the detection of matrix metalloproteases (MMPs) were used to evaluate the system. Diffusion-rate limited solid phase reactions were enhanced by microagitation using the SAW technology resulting in up to three-fold higher signals.

Keywords: Automation; Surface acoustic waves (SAW); Microagitation; Protein microarrays; Diffusion limitation

Increasing robustness and sensitivity of protein microarrays through microagitation and automation by M. Hartmann; A. Toegl; R. Kirchner; M.F. Templin; T.O. Joos (pp. 66-73).

Keywords: Automation; Surface acoustic waves (SAW); Microagitation; Protein microarrays; Diffusion limitation

Advances in cancer tissue microarray technology: Towards improved understanding and diagnostics by Wenjin Chen; David J. Foran (pp. 74-81).

Over the past few years, tissue microarray (TMA) technology has been established as a standard method for assessing the expression of proteins or genes across large sets of tissue specimens. It is being adopted increasingly among leading research institutions around the world and utilized in cancer research in parallel with the cDNA microarray technology. This article summarizes various aspects of cancer understanding and diagnostics in which TMA has had great impact. Although tremendous advances continue to be made to facilitate imaging and archiving of TMA specimens, automatic evaluation and quantitative analysis of TMA still remains an important challenge for modern investigators.

Keywords: Tissue microarray; Molecular profiling; Molecular marker quantification; Automatic tissue analysis

Advances in cancer tissue microarray technology: Towards improved understanding and diagnostics by Wenjin Chen; David J. Foran (pp. 74-81).

Keywords: Tissue microarray; Molecular profiling; Molecular marker quantification; Automatic tissue analysis

Optimization of aptamer microarray technology for multiple protein targets by Eun Jeong Cho; James R. Collett; Anna E. Szafranska; Andrew D. Ellington (pp. 82-90).

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5mM MgCl₂ and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72pg/mL (5pM, lysozyme), 15ng/mL (0.5nM, ricin), 1.9ng/mL (0.01nM, IgE), and 170ng/mL (5nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.

Keywords: Aptamer; Microarray; Protein; Multiplex; Proteomics

Optimization of aptamer microarray technology for multiple protein targets by Eun Jeong Cho; James R. Collett; Anna E. Szafranska; Andrew D. Ellington (pp. 82-90).

Keywords: Aptamer; Microarray; Protein; Multiplex; Proteomics

Selection of aptamers by systematic evolution of ligands by exponential enrichment: Addressing the polymerase chain reaction issue by Michael U. Musheev; Sergey N. Krylov (pp. 91-96).

Aptamers are DNA oligonucleotides capable of binding different classes of targets with high affinity and selectivity. They are particularly attractive as affinity probes in multiplexed quantitative analysis of proteins. Aptamers are typically selected from large libraries of random DNA sequences in a general approach termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX involves repetitive rounds of two processes: (i) partitioning of aptamers from non-aptamers by an affinity method and (ii) amplification of aptamers by the polymerase chain reaction (PCR). New partitioning methods, which are characterized by exceptionally high efficiency of partitioning, have been recently introduced. For the overall SELEX procedure to be efficient, the high efficiency of new partitioning methods has to be matched by high efficiency of PCR. Here we present the first detailed study of PCR amplification of random DNA libraries used in aptamer selection. With capillary electrophoresis as an analytical tool, we found fundamental differences between PCR amplification of homogeneous DNA templates and that of large libraries of random DNA sequences. Product formation for a homogeneous DNA template proceeds until primers are exhausted. For a random DNA library as a template, product accumulation stops when PCR primers are still in excess of the products. The products then rapidly convert to by-products and virtually disappear after only 5 additional cycles of PCR. The yield of the products decreases with the increasing length of DNA molecules in the library. We also proved that the initial number of DNA molecules in PCR mixture has no effect on the by-products formation. While the increase of the Taq DNA polymerase concentration in PCR mixture selectively increases the yield of PCR products. Our findings suggest that standard procedures of PCR amplification of homogeneous DNA samples cannot be transferred to PCR amplification of random DNA libraries: to ensure efficient SELEX, PCR has to be optimized for the amplification of random DNA libraries.

Keywords: Abbreviations; CE; capillary electrophoresis; PCR; polymerase chain reaction; SELEX; systematic evolution of ligands by exponential enrichment; NECEEM; non-equilibrium capillary electrophoresis of equilibrium mixtures; ssDNA; single-stranded DNA; dsDNA; double-stranded DNA; ss-dsDNA; DNA with both single-stranded and double-stranded regions; FAM; 6-carboxyfluorescein; dNTP; deoxyribonucleotide triphosphates; SSB; single-stranded binding proteinAptamers; PCR; DNA library; Capillary electrophoresis; Protein Analysis; SELEX

Selection of aptamers by systematic evolution of ligands by exponential enrichment: Addressing the polymerase chain reaction issue by Michael U. Musheev; Sergey N. Krylov (pp. 91-96).

Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations by Ruin Moaddel; Irving W. Wainer (pp. 97-105).

Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities ( K_d values) and non-linear chromatography can be used to assess the association ( k_on) and dissociation ( k_off) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

Keywords: Nicotinic receptors; Drug transporters; Affinity chromatography; Displacement chromatography

Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases by Travis R. Besanger; Richard J. Hodgson; James R.A. Green; John D. Brennan (pp. 106-115).

Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol–gel processing route. While such columns proved to be useful for applications such as screening of protein–ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000nL/min) and backpressure generated (from 500 to 2100psi) during the chromatographic activity assay owing to increases in k_cat and decreases in K_M, switching from diffusion controlled to reaction controlled conditions at ca. 2000psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography.

Keywords: Immobilized enzyme reactor chromatography; Protein retention; Enzyme activity

Protein digestion and phosphopeptide enrichment on a glass microchip by Guihua Eileen Yue; Michael G. Roper; Catherine Balchunas; Abigail Pulsipher; Joshua J. Coon; Jeffery Shabanowitz; Donald F. Hunt; James P. Landers; Jerome P. Ferrance (pp. 116-122).

This work describes an integrated glass microdevice for proteomics, which directly couples proteolysis with affinity selection. Initial results with standard phosphopeptide fragments from β-casein in peptide mixtures showed selective capture of the phosphorylated fragments using immobilized metal affinity chromatography (IMAC) beads packed into a microchannel. Complete selectivity was seen with angiotensin, with capture of only the phosphorylated form. On-chip proteolysis, using immobilized trypsin beads packed into a separate channel, was directly coupled to the phosphopeptide capture and the integrated devices evaluated using β-casein. Captured and eluted fragments were analyzed using both capillary electrophoresis (CE) and capillary liquid chromatography/mass spectrometry (cLC/MS). The results show selective capture of only phosphopeptide fragments, but incomplete digestion of the protein was apparent from multiple peaks in the CE separations. The MS analysis indicated a capture bias on the IMAC column for the tetraphosphorylated peptide fragment over the monophosphorylated fragment. Application to digestion and capture of a serum fraction showed capture of material; however, non-specific binding was evident. Additional work will be required to fully optimize this system, but this work represents a novel sample preparation method, incorporating protein digestion on-line with affinity capture for proteomic applications.

Keywords: Microchip; Phosphopeptide; Mass spectrometry; Protein digestion; IMAC

Creating hierarchical models of protein families based on Expressed Sequence Tags: The “Sprockets�? analysis pipeline by Paul M.K. Gordon; Christian Weinel; Carsten Jacobi; Udo Kämpf; Evgenia Kriventseva; Christoph W. Sensen (pp. 123-132).

We have created an analysis pipeline called Sprockets, which can be used to classify proteins into various hierarchical “families�?, and build searchable models of these families. The construction of these families is based on data from Expressed Sequence Tags (ESTs) and Coding DNA Sequences (CDSs), making Sprockets clusters especially suitable for studying gene families in organisms for which the completely sequenced genome does not (yet) exist. The pipeline consists of two main parts: pair-wise analysis and grouping of sequences with Z-score statistics, followed by hierarchical splitting of clusters into alignable protein families. Various computational and statistical techniques applied in Sprockets allow it to act like a massive and selective multiple sequence alignment engine for combining individual sequence collections and related public sequences. The end result is a database of gene Hidden Markov Models, each related to the other by three levels of similarity: secondary structure, function and evolutionary origin. For a sample 20,000 EST set from Lactuca spp., Sprockets provided a 9% improvement in mapping of function to unknown sequences over traditional pair-wise search methods and InterPro mapping.

Keywords: EST assembly; Protein families; Hidden Markov Models; Sequence clustering; Multiple sequence alignments; Sprockets

Creating hierarchical models of protein families based on Expressed Sequence Tags: The “Sprockets” analysis pipeline by Paul M.K. Gordon; Christian Weinel; Carsten Jacobi; Udo Kämpf; Evgenia Kriventseva; Christoph W. Sensen (pp. 123-132).

We have created an analysis pipeline called Sprockets, which can be used to classify proteins into various hierarchical “families”, and build searchable models of these families. The construction of these families is based on data from Expressed Sequence Tags (ESTs) and Coding DNA Sequences (CDSs), making Sprockets clusters especially suitable for studying gene families in organisms for which the completely sequenced genome does not (yet) exist. The pipeline consists of two main parts: pair-wise analysis and grouping of sequences with Z-score statistics, followed by hierarchical splitting of clusters into alignable protein families. Various computational and statistical techniques applied in Sprockets allow it to act like a massive and selective multiple sequence alignment engine for combining individual sequence collections and related public sequences. The end result is a database of gene Hidden Markov Models, each related to the other by three levels of similarity: secondary structure, function and evolutionary origin. For a sample 20,000 EST set from Lactuca spp., Sprockets provided a 9% improvement in mapping of function to unknown sequences over traditional pair-wise search methods and InterPro mapping.

Keywords: EST assembly; Protein families; Hidden Markov Models; Sequence clustering; Multiple sequence alignments; Sprockets

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Analytica Chimica Acta (v.564, #1)