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Sequence Coverage (sequence + coverage)
Selected AbstractsHPTLC/DESI-MS imaging of tryptic protein digests separated in two dimensions,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2008Sofie P. Pasilis Abstract Desorption electrospray ionization mass spectrometry (DESI-MS) was demonstrated as a method to detect and identify peptides from two-dimensional separations of cytochrome c and myoglobin tryptic digests on ProteoChrom HPTLC Cellulose sheets. Data-dependent tandem mass spectra were acquired during lane scans across the TLC plates. Peptides and the corresponding proteins were identified using a protein database search software. Two-dimensional distributions of identified peptides were mapped for each separated protein digest. Sequence coverages for cytochrome c and myoglobin were 81 and 74%, respectively. These compared well with those determined using the more standard HPLC/ESI-MS/MS approach (89 and 84%, respectively). Preliminary results show that use of more sensitive instrumentation has the potential for improved detection of peptides with low Rf values and improvement in sequence coverage. However, less multiple charging and more sodiation were seen in HPTLC/DESI-MS spectra relative to HPLC/ESI-MS spectra, which can affect peptide identification by MS/MS. Methods to increase multiple charging and reduce the extent of sodiation are currently under investigation. Published in 2008 by John Wiley & Sons, Ltd. [source] Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampusELECTROPHORESIS, Issue 11 2010Seok Heo Abstract Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate,aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. ModiroÔ v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease. [source] A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysisELECTROPHORESIS, Issue 23 2007Hu Zhou Abstract The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome. [source] High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometryELECTROPHORESIS, Issue 21 2003Alexander R. Ivanov Abstract High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 ,m inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n -propanol and formamide as porogens and azobisisobutyronitrile as initiator. N -Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300,000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method. [source] Comparison of automated in-gel digest methods for femtomole level samplesELECTROPHORESIS, Issue 19-20 2003Erin J. Finehout Abstract A comparison of automated in-gel digestion methods for low picomolar to femtomolar levels of protein is presented. Gel spots with 4 pmol to 120 fmol of protein were stained with either Coomassie colloidal blue or SYPRO Ruby and digested using an automated platform. The sequence coverages and average peak intensities obtained from a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis are compared. Results show that methods using an acetonitrile extraction or digest times greater than the standard 4 h give no significant increase in peptide sequence coverage for automated digestion of low protein level samples. It is also shown that digests from SYPRO Ruby-stained gels show a greater improvement upon ZipTip cleanup than digests from Coomassie colloidal blue-stained gels. The digests from SYPRO Ruby-stained gels are also shown to give a higher average peptide intensity if a method with minimal gel plug washing is used. [source] Functionalization Strategies for Protease Immobilization on Magnetic NanoparticlesADVANCED FUNCTIONAL MATERIALS, Issue 11 2010Dan Li Abstract A comprehensive study on the general functionalization strategies for magnetic nanoparticles (MNPs) is presented in this work. Using well-established techniques as well as modified protocols, the wide range of functional moieties grafted on ,-Fe2O3 (maghemite) nanosurfaces include those of amine, aldehyde, carboxylic, epoxy, mercapto, and maleimide ends. Among the modified protocols are the one-step water-catalyzed silanization with mercaptopropyltrimethoxysilane, resulting in dense distal thiols, and the direct functionalization with a heterogeneous bifunctional linker N -[p-maleimidophenyl]isocynanate (PMPI). The former results in a protective Stöber type coating while simultaneously reducing the iron oxide core to magnetite (Fe3O4). The conjugation of trypsin, hereby chosen as model biomolecule, onto the differently functionalized MNPs is further demonstrated and assessed based on its activity, kinetics, and thermo-/long-term stability as well as reusability. Besides aqueous stability and ease in recovery by magnetic separation, the immobilized trypsin on MNPs offers superior protease durability and reusability, without compromising the substrate specificity and sequence coverage of free trypsin. The MNP-based proteases can be used as valuable carriers in proteomics and miniaturized total analysis devices. The applicability of the functional surfaces devised in the current study is also relevant for the conjugation of other biomolecules beyond trypsin. [source] HPTLC/DESI-MS imaging of tryptic protein digests separated in two dimensions,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2008Sofie P. Pasilis Abstract Desorption electrospray ionization mass spectrometry (DESI-MS) was demonstrated as a method to detect and identify peptides from two-dimensional separations of cytochrome c and myoglobin tryptic digests on ProteoChrom HPTLC Cellulose sheets. Data-dependent tandem mass spectra were acquired during lane scans across the TLC plates. Peptides and the corresponding proteins were identified using a protein database search software. Two-dimensional distributions of identified peptides were mapped for each separated protein digest. Sequence coverages for cytochrome c and myoglobin were 81 and 74%, respectively. These compared well with those determined using the more standard HPLC/ESI-MS/MS approach (89 and 84%, respectively). Preliminary results show that use of more sensitive instrumentation has the potential for improved detection of peptides with low Rf values and improvement in sequence coverage. However, less multiple charging and more sodiation were seen in HPTLC/DESI-MS spectra relative to HPLC/ESI-MS spectra, which can affect peptide identification by MS/MS. Methods to increase multiple charging and reduce the extent of sodiation are currently under investigation. Published in 2008 by John Wiley & Sons, Ltd. [source] MassSieve: Panning MS/MS peptide data for proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2010Douglas J. Slotta Abstract We present MassSieve, a Java-based platform for visualization and parsimony analysis of single and comparative LC-MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC-MS/MS-based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments. [source] Application of electron transfer dissociation (ETD) for the analysis of posttranslational modificationsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 21 2008Julia Wiesner Abstract Despite major advantages in the field of proteomics, the analysis of PTMs still poses a major challenge; thus far, preventing insights into the role and regulation of protein networks. Additionally, top-down sequencing of proteins is another powerful approach to reveal comprehensive information for biological function. A commonly used fragmentation technique in MS-based peptide sequencing is CID. As CID often fails in PTM-analysis and performs best on doubly-charged, short and middle-sized peptides, confident peptide identification may be hampered. A newly developed fragmentation technique, namely electron transfer dissociation (ETD), supports both, PTM- and top-down analysis, and generally results in more confident identification of long, highly charged or modified peptides. The following review presents the theoretical background of ETD and its technical implementation in mass analyzers. Furthermore, current improvements of ETD and approaches for the PTM-analysis and top-down sequencing are introduced. Alternating both fragmentation techniques, ETD and CID, increases the amount of information derived from peptide fragmentation, thereby enhancing both, peptide sequence coverage and the confidence of peptide and protein identification. [source] On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 20 2007Yan Li Abstract In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5,min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12,h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins. [source] MapQuant: Open-source software for large-scale protein quantificationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Kyriacos C. Leptos Abstract Whole-cell protein quantification using MS has proven to be a challenging task. Detection efficiency varies significantly from peptide to peptide, molecular identities are not evident a,priori, and peptides are dispersed unevenly throughout the multidimensional data space. To overcome these challenges we developed an open-source software package, MapQuant, to quantify comprehensively organic species detected in large MS datasets. MapQuant treats an LC/MS experiment as an image and utilizes standard image processing techniques to perform noise filtering, watershed segmentation, peak finding, peak fitting, peak clustering, charge-state determination and carbon-content estimation. MapQuant reports abundance values that respond linearly with the amount of sample analyzed on both low- and high-resolution instruments (over a 1000-fold dynamic range). Background noise added to a sample, either as a medium-complexity peptide mixture or as a high-complexity trypsinized proteome, exerts negligible effects on the abundance values reported by MapQuant and with coefficients of variance comparable to other methods. Finally, MapQuant's ability to define accurate mass and retention time features of isotopic clusters on a high-resolution mass spectrometer can increase protein sequence coverage by assigning sequence identities to observed isotopic clusters without corresponding MS/MS data. [source] Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: The human mammary epithelial cell proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Tao Liu Abstract Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14,416 confidently identified peptides covering 4294 different proteins with an estimated 10%,gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390,proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein Mr,, pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems. [source] Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell linesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2003Chih-Lei Lee Abstract An expression map of the most abundant proteins in human hepatoma HepG2 cells was established by a combination of complementary shotgun proteomics approaches. Two-dimensional liquid chromatography (LC)-nano electrospray ionization (ESI) tandem mass spectrometry (MS/MS) as well as one-dimensional LC-matrix-assisted laser desorption/ionization MS/MS were evaluated and shown that additional separation introduced at the peptide level was not as efficient as simple prefractionation of protein extracts in extending the range and total number of proteins identified. Direct LC-nanoESI MS/MS analyses of peptides from total solubilized fraction and the excised gel bands from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated insolubilized fraction afforded the best combination in efficient construction of a nonredundant cell map. Compiling data from multiple variations of rapid shotgun proteomics analyses is nonetheless useful to increase sequence coverage and confidence of hits especially for those proteins identified primarily by a single or two peptide matches. While the returned hit score in general reflects the abundance of the respective proteins, it is not a reliable index for differential expression. Using another closely related hepatoma Hep3B as a comparative basis, 16 proteins with more than two-fold difference in expression level as defined by spot intensity in two-dimensional gel electrophoresis analysis were identified which notably include members of the heat shock protein (Hsp) and heterogeneous nuclear ribonucleoprotein (hnRPN) families. The observed higher expression level of hnRNP A2/B1 and Hsp90 in Hep3B led to a search for reported functional roles mediated in concert by both these multifunctional cellular chaperones. In agreement with the proposed model for telomerase and telomere bound proteins in promoting their interactions, data was obtained which demonstrated that the expression proteomics data could be correlated with longer telomeric length in tumorigenic Hep3B. This biological significance constitutes the basis for further delineation of the dynamic interactions and modifications of the two protein families and demonstrated how proteomic and biological investigation could be mutually substantiated in a productive cycle of hypothesis and pattern driven research. [source] Assessment of acetone as an alternative to acetonitrile in peptide analysis by liquid chromatography/mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2009Ria Fritz Acetonitrile as a solvent used in liquid chromatography/mass spectrometry (LC/MS) of peptides and proteins is a relatively toxic solvent (LD50 oral; rat; 2,460,mg/kg) compared to alternatives like methanol (LD50 oral; rat; 5,628,mg/kg) and acetone (LD50 oral; rat; 5,800,mg/kg). Strategies to minimize its consumption in LC are either to reduce the inner diameter of the column or replace acetonitrile with a suitable alternative. Methanol is often recommended to replace acetonitrile in peptide analysis. In this study however, the main focus lies on another alternative solvent for LC/MS of peptides; acetone. A number of model proteins were tryptically digested and the peptide solutions were analyzed on a linear trap quadrupole (LTQ) mass spectrometer. The performances of acetonitrile, methanol and acetone were compared according to the quality of the chromatograms obtained and identification of the peptides using the BioWorksÔ software developed by Thermo Scientific. In accordance to the elutropic series, acetone was found to significantly reduce the retention times of peptides separated by C18 column material with regard to acetonitrile while methanol led to increased retention times. Acetone was the superior solvent to methanol for most of the tested model proteins reaching similar sequence coverage and numbers of identified peptides as acetonitrile. We therefore propose acetone as an alternative to acetonitrile in LC/MS of peptides. Copyright © 2009 John Wiley & Sons, Ltd. [source] Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow regionRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2009T. Yu. A high-performance liquid chromatography nano-electrospray ionization Fourier transform mass spectrometry (HPLC/nanoESI-FTMS) approach involving recording of collision-activated dissociation (CAD) and electron-capture dissociation (ECD) spectra of an intact sample and two its modifications after performic oxidation and reduction followed by carboxamidomethylation helps to establish peptide profiles in the crude secretion of frog species at mid-throughput level, including de novo sequencing. The proposed derivatization procedures allow increasing of the general sequence coverage in the backbone, providing complementary information and, what is more important, reveal the amino acid sequence in the cystine ring (,rana box'). Thus purely mass spectrometric efficient sequencing becomes possible for longer than usual proteolytic peptides. Seventeen peptides belonging to four known families were identified in the secretion of the European brown frog Rana arvalis inhabiting the Moscow region in Russia. Ranatuerins, considered previously a unique feature of the North American species, as well as a new melittin-related peptide, are worth special mention. The developed approach was previously successfully used for the identification of peptides in the skin secretion of the Caucasian green frog Rana ridibunda. Copyright © 2009 John Wiley & Sons, Ltd. [source] Top-down proteomics with a quadrupole time-of-flight mass spectrometer and collision-induced dissociationRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2009Andrea Armirotti With slight modifications of the instrumental parameters, we demonstrate that satisfactory top-down data can be obtained with collision-induced dissociation (CID) tandem mass spectrometry on a quadrupole time-of-flight (qTOF) instrument not originally designed for this purpose. Protein identification is achieved with both N- and C-terminal sequence tags and BLAST database searches. The accurate mass measurement of multiply charged fragment ions (mostly y and b-type) supplements the limited set of cleavage sites and provides a high degree of sequence coverage (90,100%). Post-translational modification issues can be addressed too. This approach might help those mass spectrometry (MS) core facilities that are not able to afford very high-resolution instruments, thus expanding the benefits of top-down protein analysis over the worldwide MS community. Copyright © 2009 John Wiley & Sons, Ltd. [source] Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2009William R. Alley Jr. Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip-based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C18 LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C18 chips. On the other hand, hydrophobic glycopeptides were better retained on C18 chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1,pmol). The performance of both media also appeared comparable when analyzing a four-protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase. Copyright © 2009 John Wiley & Sons, Ltd. [source] In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2006Brigitte L. Simons We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100,mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the , -amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b -ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9 - to D9 -trimethyl-labeled peptides of , -casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes. Published in 2006 by John Wiley & Sons, Ltd. [source] Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2006Mariola Batycka Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500,fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9,min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30,min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) in combination with LC/MS/MS using 4,min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of post-translational modifications (PTMs). Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS-MS/MS switching and scanning capabilities. Copyright © 2006 John Wiley & Sons, Ltd. [source] Capillary liquid chromatography/atmospheric-pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry: a comparison with liquid chromatography/matrix-assisted laser desorption/ionisation time-of-flight and liquid chromatography/electrospray ionisation quadrupole time-of-flight for the identification of tryptic peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2006Colin S. Creaser The atmospheric-pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI-QIT) analysis of tryptic peptides is reported following capillary liquid chromatographic (LC) separation and direct analysis of a protein digest. Peptide fragments were identified by peptide mass fingerprinting from mass spectrometric data and sequence analysis obtained by tandem mass spectrometry of the principal mass spectral peaks using a data-dependent scanning protocol. These data were compared with those from mass spectrometric analysis using capillary LC/MALDI-time-of-flight (TOF) and capillary LC/electrospray ionisation (ESI)-quadrupole TOF. For all three configurations the resulting data were searched against the MSDB database, using MASCOT and the sequence coverage compared for each technique. Complementary data were obtained using the three techniques. Copyright © 2006 John Wiley & Sons, Ltd. [source] Fragmentation of oligosaccharide ions with 157,nm vacuum ultraviolet lightRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2005Arugadoss Devakumar The 157,nm photofragmentation of native and derivatized oligosaccharides was studied in a linear ion trap and in a home-built matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometer, and the results were compared with collision-induced dissociation (CID) experiments. Photodissociation produces product ions corresponding to high-energy fragmentation pathways; for cation-derivatized oligosaccharides, it yields strong cross-ring fragment ions and provides better sequence coverage than low- and high-energy CID experiments. On the other hand, for native oligosaccharides, CID yielded somewhat better sequence coverage than photodissociation. The ion trap enables CID hybrid MS3 experiments on the high-energy fragment ions obtained from photodissociation. Copyright © 2005 John Wiley & Sons, Ltd. [source] Automating proteome analysis: improvements in throughput, quality and accuracy of protein identification by peptide mass fingerprinting,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2004Ludovic Canelle The use of robots has major effects on maximizing the proteomic workflow required in an increasing number of high-throughput projects and on increasing the quality of the data. In peptide mass finger printing (PMF), automation of steps downstream of two-dimensional gel electrophoresis is essential. To achieve this goal, the workflow must be fluid. We have developed tools using macros written in Microsoft Excel and Word to complete the automation of our platform. Additionally, because sample preparation is crucial for identification of proteins by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we optimized a sandwich method usable by any robot for spotting digests on a MALDI target. This procedure enables further efficient automated washing steps directly on the MALDI target. The success rate of PMF identification was evaluated for the automated sandwich method, and for the dried-droplet method implemented on the robot as recommended by the manufacturer. Of the two methods, the sandwich method achieved the highest identification success rate and sequence coverage of proteins. Copyright © 2004 John Wiley & Sons, Ltd. [source] Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteinsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2003Laetitia Cravello The combination of hydrogen exchange and mass spectrometry has been widely used in structural biology, providing views on protein structure and protein dynamics. One of the constraints is to use proteases working at low pH and low temperature to limit back-exchange during proteolysis. Although pepsin works in these conditions and is currently used in such experiments, sequence coverage is not always complete especially for large proteins, and the spatial resolution of the exchange rate is limited by the size of the resulting peptides. In this study we tried two other proteases, protease type XIII from Aspergillus saitoi and protease type XVIII from Rhizhopus species. The penicillin-binding protein X (PBP-2X*), a 77-kDa protein, was selected as a model. Like pepsin, neither of these proteases is really specific, but we found very good reproducibility in the digestion pattern. Compared with using pepsin alone, combining the results of the three independent proteolyses increased the coverage for the peptide mapping, thus avoiding missing some potentially interesting regions of the protein. Furthermore, we obtained a better spatial resolution for deuterium incorporation data, specifying accurately the deuterated regions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Reactivity of the NS2/3(907,1206)ASK4 protein with ,-mercaptoethanol studied by electrospray ion trap mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2002Laura Orsatti The present work reports a mass spectrometric investigation of the NS2/3 protein, a protease from hepatitis C virus (HCV). During routine protein manipulation, in the presence of 100,mM ,-mercaptoethanol and under denatured conditions, the protein was unexpectedly modified at its cysteine residues, and the increased molecular weight corresponded to one molecule of ,-mercaptoethanol bound. The modified protein, once refolded, was found to be less active than the unmodified one. The aim of this work was to investigate whether the reactivity of cysteines with ,-mercaptoethanol involves one specific, highly reactive residue of the sequence, or if the modification is a random process. Liquid chromatography (LC) coupled on-line with an electrospray ion trap mass spectrometer was used to identify the modification sites. It was found that five cysteines out of nine had reacted with ,-mercaptoethanol, none of them showing a significantly higher reactivity than the others. 95% of sequence coverage was obtained. Copyright © 2002 John Wiley & Sons, Ltd. [source] Micro-high-performance liquid chromatography/Fourier transform mass spectrometry with electron-capture dissociation for the analysis of protein enzymatic digestsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2002Walter Davidson Electron-capture dissociation (ECD) Fourier transform mass spectrometry (FTMS) employed to generate comprehensive sequence information for the chromatographic analysis of enzymatic protein digests is described. A pepsin digest of cytochrome c was separated by reversed-phase micro-high-performance liquid chromatography (µHPLC) and ionized ,on-line' by electrospray ionization (ESI). The ions thus formed were transferred to and trapped in the FTMS analyzer cell. Typically, no precursor ion isolation was performed. The trapped ions were subjected to a pulse of electrons to induce fragmentation. Mass spectra were acquired continuously to produce a three-dimensional LC/MS data set. The spectra were dominated by c and, to a lesser degree, z ions, which provided near complete sequence coverage. External calibration provided good mass accuracy and resolution, typical of FTMS. Thus,µHPLC/ECD,,,FTMS is shown to be a highly informative method for the analysis of enzymatic protein digests. Copyright © 2002 John Wiley & Sons, Ltd. [source] Factors determining the performance of triple quadrupole, quadrupole ion trap and sector field mass spectrometer in electrospray ionization mass spectrometry.RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2001The sequence coverage by fragment ions resulting from collision-induced dissociation in a triple stage quadrupole (TSQ) and a quadrupole ion trap (QIT) mass spectrometer of 10,20-mer oligonucleotides was investigated. While (a-B) and w ion series were the most abundant on both instruments, additional ion series of sequence relevance were preferably formed in the TSQ. Thus, a total number of 83 fragment ions were used to deduce the complete sequence of a 10-mer oligonucleotide of mixed sequence from a tandem mass spectrum recorded on the TSQ. The complete sequence was also encoded in the 28 fragments that were obtained from the QIT under comparable fragmentation conditions. Spectrum complexity increased considerably at the cost of signal-to-noise ratio upon fragmentation of a 20-mer oligonucleotide in the TSQ, whereas spectrum interpretation with longer oligonucleotides was significantly more straightforward in spectra recorded on the QIT. The extent of fragmentation had to be optimized by appropriate setting of collision energy and choice of precursor ion charge state in order to obtain full sequence coverage by fragments for de novo sequencing. Moreover, full sequence information was also dependent on base sequence because of the low tendency of backbone cleavage at thymidines. Tandem mass spectrometry on the QIT yielded redundant information that was successfully utilized to deduce the complete sequence of 20-mer oligonucleotides with high confidence. Copyright © 2001 John Wiley & Sons, Ltd. [source] Combining Microarray-based Genomic Selection (MGS) with the Illumina Genome Analyzer Platform to Sequence Diploid Target RegionsANNALS OF HUMAN GENETICS, Issue 5 2009David T. Okou Summary Novel methods of targeted sequencing of unique regions from complex eukaryotic genomes have generated a great deal of excitement, but critical demonstrations of these methods efficacy with respect to diploid genotype calling and experimental variation are lacking. To address this issue, we optimized microarray-based genomic selection (MGS) for use with the Illumina Genome Analyzer (IGA). A set of 202 fragments (304 kb total) contained within a 1.7 Mb genomic region on human chromosome X were MGS/IGA sequenced in ten female HapMap samples generating a total of 2.4 GB of DNA sequence. At a minimum coverage threshold of 5X, 93.9% of all bases and 94.9% of segregating sites were called, while 57.7% of bases (57.4% of segregating sites) were called at a 50X threshold. Data accuracy at known segregating sites was 98.9% at 5X coverage, rising to 99.6% at 50X coverage. Accuracy at homozygous sites was 98.7% at 5X sequence coverage and 99.5% at 50X coverage. Although accuracy at heterozygous sites was modestly lower, it was still over 92% at 5X coverage and increased to nearly 97% at 50X coverage. These data provide the first demonstration that MGS/IGA sequencing can generate the very high quality sequence data necessary for human genetics research. All sequences generated in this study have been deposited in NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra, Accession # SRA007913). [source] On the use of ESI-QqTOF-MS/MS for the comparative sequencing of nucleic acidsBIOPOLYMERS, Issue 6 2009Herbert Oberacher Abstract The usability of a quadrupole,quadrupole,time-of-flight (QqTOF) instrument for the tandem mass spectrometric sequencing of oligodeoxynuleotides was investigated. The sample set consisted of 21 synthetic oligodeoxynucleotides ranging in length from 5 to 42 nucleotides. The sequences were randomly selected. For the majority of tested oligonucleotides, two or three different charge states were selected as precursor ions. Each precursor ion was fragmented applying several different collision voltages. Overall 282 fragment ion mass spectra were acquired. Computer-aided interpretation of fragment ion mass spectra was accomplished with a recently introduced comparative sequencing algorithm (COMPAS). The applied version of COMPAS was specifically optimized for the interpretation of information-rich spectra obtained on the QqTOF. Sequences of oligodeoxynucleotides as large as 26-mers were correctly verified in >94% of cases (182 of 192 spectra acquired). Fragment ion mass spectra of larger oligonucleotides were not specific enough for sequencing. Because of the occurrence of extensive internal fragmentation causing low sequence coverage paired with a high probability of assigning fragment ions to wrong sequences, tandem mass spectra obtained from oligonucleotides consisting of 30 and more nucleotides could not be used for sequence verification neither manually nor with COMPAS. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 401,409, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Comparison of two glutaraldehyde immobilization techniques for solid-phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometryELECTROPHORESIS, Issue 9 2004Isabelle Migneault Abstract Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively. [source] Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometerRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 3 2008Desmond A. Kaplan Electron transfer dissociation (ETD) of proteins is demonstrated in a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer (Qh-FTICRMS). Analyte ions are selected in the mass analyzing quadrupole, accumulated in the hexapole linear ion trap, reacted with fluoranthene reagent anions, and then analyzed via an FTICR mass analyzer. The hexapole trap allows for a broad fragment ion mass range and a high ion storage capacity. Using a 3,T FTICRMS, resolutions of 60,000 were achieved with mass accuracies averaging below 1.4,ppm. The high resolution, high mass accuracy ETD spectra provided by FTICR obviates the need for proton transfer reaction (PTR) charge state reduction of ETD product ions when analyzing proteins or large peptides. This is demonstrated with the ETD of ubiquitin and apomyoglobin yielding sequence coverages of 37 and 20%, respectively. We believe this represents the first reported successful combination of ETD and a FTICRMS. Copyright © 2008 John Wiley & Sons, Ltd. [source] | |||||||||||||