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Thomas A Neubert - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Electroblotted Proteins by Mass Spectrometry.
Methods of Molecular Biology, 2020Co-Authors: Jose L. Luque-garcia, Thomas A NeubertAbstract:Identification of proteins by mass spectrometry is crucial for better understanding of many biological, biochemical, and biomedical processes. Here we describe two methods for the identification of electroblotted proteins by on-membrane Digestion prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of In-Gel Digestion and provide better Digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present.
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Evaluation of the Variation in Sample Preparation for Comparative Proteomics Using Stable Isotope Labeling by Amino Acids in Cell Culture
Journal of Proteome Research, 2009Co-Authors: Guoan Zhang, David Fenyö, Thomas A NeubertAbstract:In comparative proteomic studies, it is important to know the variability associated with sample preparation. In this study, we report the strategy of using SILAC (stable isotope labeling by amino acids in cell culture) to evaluate the effect of the variation in sample preparation for quantitative proteomics. Variability can be measured when equal amounts of light and heavy SILAC samples undergo the same sample preparation procedures in parallel, and the two samples are mixed for relative protein quantitation by mass spectrometry. The high quantitative accuracy of SILAC allows for characterization of small variations. First, the reproducibility of immunoprecipitation (IP) and In-Gel Digestion was evaluated, and the impact of replicate number on quantitative accuracy was characterized. Second, we evaluated the overall variation in a comparative workflow involving three sequential sample preparation steps: IP, SDS-PAGE fractionation, and In-Gel Digestion. The evaluation of individual sample preparation step...
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On-membrane tryptic Digestion of proteins for mass spectrometry analysis.
Methods of Molecular Biology, 2009Co-Authors: Jose L. Luque-garcia, Thomas A NeubertAbstract:Identification of proteins and characterization of posttranslational modifications are crucial steps for many biological, biochemical, and biomedical studies, and mass spectrometry has become the method of choice for these analyses. Here we describe two methods for the on-membrane Digestion of proteins electroblotted onto nitrocellulose membranes prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of In-Gel Digestion and provide better Digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present.
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analysis of electroblotted proteins by mass spectrometry protein identification after western blotting
Molecular & Cellular Proteomics, 2008Co-Authors: Jose L Luquegarcia, Ge Zhou, Daniel S Spellman, Thomas A NeubertAbstract:SUMMARY We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting And Removal of Nitrocellulose, or BARN), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with mass spectrometry analysis. This method offers improved protein coverage, especially for membrane proteins such as uroplakins, since the extraction step after In-Gel Digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with In-Gel Digestion.
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Use of Nitrocellulose Membranes for Protein Characterization by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
Analytical Chemistry, 2006Co-Authors: Jose L. Luque-garcia, Ge Zhou, Thomas A NeubertAbstract:We present an improved method for MALDI-MS analysis of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted proteins can be analyzed directly for intact molecular weight determination or after on-membrane Digestion by dissolution of the nitrocellulose in MALDI matrix solution containing 70% acetonitrile and 30% methanol. This solution helps maintain solubility of proteins and peptides while dissolving the NC membrane, which is dissolved by 100% acetone in other protocols. On-membrane tryptic Digestion using this method requires half the time of In-Gel Digestion and results in fewer missed cleavages and better protein coverage. For the membrane proteins studied, bovine uroplakins II and III, the protein coverage was almost twice that provided by conventional In-Gel Digestion, and the transmembrane domains of both uroplakins were detected only after on-membrane Digestion. We also demonstrated the compatibility with MALDI-MS of a new dye, MemCode, whi...
Andrej Shevchenko - One of the best experts on this subject based on the ideXlab platform.
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In-Gel Digestion for mass spectrometric characterization of proteins and proteomes
Nature Protocols, 2007Co-Authors: Andrej Shevchenko, Henrik Tomas, Jan Havliš, Jesper V. Olsen, Matthias MannAbstract:In-Gel Digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the In-Gel Digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
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Absolute Quantification of Proteins in Solutions and in Polyacrylamide Gels by Mass Spectrometry
Analytical Chemistry, 2004Co-Authors: Jan Havliš, Andrej ShevchenkoAbstract:A combination of nanoelectrospray tandem mass spectrometry and 18O-labeled peptide internal standards was applied for the absolute quantification of proteins from their in-solution and In-Gel tryptic digests. Although absolute quantification from in-solution digests was accurate, we observed that In-Gel Digestion compromised the quantification accuracy by affecting the recovery of individual peptides and, therefore, the provided estimates might be strongly influenced by the selection of reference peptides. Under optimized experimental conditions, it was possible to provide a semiquantitative estimate of the absolute amount of gel separated proteins within better than 50% error margin.
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Fast-response proteomics by accelerated In-Gel Digestion of proteins
Analytical Chemistry, 2003Co-Authors: Jan Havliš, Henrik Thomas, Marek Šebela, Andrej ShevchenkoAbstract:Kinetics of In-Gel Digestion of proteins by modified and native trypsins was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards. The effect of the temperature, enzyme concentration, Digestion time, and surface area of gel pieces on the yield of Digestion products was characterized. Based on the kinetic data, we developed a protocol that enabled the identification of gel-separated proteins with 30-min Digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage. The accelerated Digestion protocol was tested in identification of more than 120 proteins from budding and fission yeasts at the subpicomole level.
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Evaluation of the efficiency of In-Gel Digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry
Analytical Biochemistry, 2001Co-Authors: Anna Shevchenko, Andrej ShevchenkoAbstract:Handbook of Microalgal Culture is truly a landmark publication, drawing on some 50 years of worldwide experience in microalgal mass culture. This important book comprises comprehensive reviews of the current available information on microalgal culture, written by 40 contributing authors from around the globe.The book is divided into four parts, with Part I detailing biological and environmental aspects of microalgae with reference to microalgal biotechnology and Part II looking in depth at major theories and techniques of mass cultivation. Part III comprises chapters on the economic applications of microalgae, including coverage of industrial production, the use of microalgae in human and animal nutrition and in aquaculture, in nitrogen fixation, hydrogen and methane production, and in bioremediation of polluted water. Finally, Part IV looks at new frontiers and includes chapters on genetic engineering, microalgae as platforms for recombinant proteins, bioactive chemicals, heterotrophic production, microalgae as gene-delivery systems for expressing mosquitocidal toxins and the enhancement of marine productivity for climate stabilization and food security.Handbook of Microalgal Culture is an essential purchase for all phycologists and also those researching aquatic systems, aquaculture and plant sciences. There is also much of great use to researchers and those involved in product formulation within pharmaceutical, nutrition and food companies. Libraries in all universities and research establishments teaching and researching in chemistry, biological and pharmaceutical sciences, food sciences and nutrition, and aquaculture will need copies of this book on their shelves.Amos Richmond is at the Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Israel.
Bernhard Granvogl - One of the best experts on this subject based on the ideXlab platform.
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Sample preparation by In-Gel Digestion for mass spectrometry-based proteomics
Analytical and Bioanalytical Chemistry, 2007Co-Authors: Bernhard Granvogl, Matthias Plöscher, Lutz Andreas EichackerAbstract:The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by “In-Gel Digestion” has been developed. Many procedures are available for In-Gel Digestion, which inspired us to review In-Gel Digestion approaches. Figure Classical In-Gel Digestion process for a protein band stained with CBB. Protein bands are cut from the polyacrylamide gel ( 1 ). CBB molecules ( blue circles ) bound to the protein are released by iterative incubation in a buffered organic solvent system ( 2 ). To increase Digestion efficiency and sequence coverage proteins are reduced ( 3 ) and alkylated ( 4 ). Proteins are subsequently digested with proteolytic enzymes ( scissors symbols ), typically trypsin ( 5 ). Trypsin cleaves at the amino acid residues arginine ( R ) and lysine ( K ). The resulting peptides ( A , B , and C ) are extracted from the polyacrylamide matrix ( 6 ). The peptide solution can be further purified for analysis by mass spectrometry (Section “ Concentration and desalting of peptides ”)
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Sample preparation by In-Gel Digestion for mass spectrometry-based proteomics.
Analytical and Bioanalytical Chemistry, 2007Co-Authors: Bernhard Granvogl, Matthias Plöscher, Lutz A. EichackerAbstract:The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by “In-Gel Digestion” has been developed. Many procedures are available for In-Gel Digestion, which inspired us to review In-Gel Digestion approaches.
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Standardisation of rapid in‐gel Digestion by mass spectrometry
Proteomics, 2007Co-Authors: Bernhard Granvogl, Patrick Gruber, Lutz A. EichackerAbstract:: In-Gel Digestion has been standardised using a poly(propylene) disposable. We designed a four-step rapid and simple In-Gel Digestion protocol which is carried out in one self-contained reaction tube avoiding keratin contamination. In order to quantify the efficiency of In-Gel Digestion, we developed a rapid on-column peptide acetylation protocol. Results show that trypsin In-Gel uptake is increased and In-Gel Digestion is 90% complete within 15 min. We further show that spectrum quality, peptide yield and sequence coverage for mass spectrometric analysis are enhanced. We utilise 2-D PAGE separation of photosystem II from barley to demonstrate that the protocol facilitates identification of highly hydrophobic membrane proteins.
Lutz Andreas Eichacker - One of the best experts on this subject based on the ideXlab platform.
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Sample preparation by In-Gel Digestion for mass spectrometry-based proteomics
Analytical and Bioanalytical Chemistry, 2007Co-Authors: Bernhard Granvogl, Matthias Plöscher, Lutz Andreas EichackerAbstract:The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by “In-Gel Digestion” has been developed. Many procedures are available for In-Gel Digestion, which inspired us to review In-Gel Digestion approaches. Figure Classical In-Gel Digestion process for a protein band stained with CBB. Protein bands are cut from the polyacrylamide gel ( 1 ). CBB molecules ( blue circles ) bound to the protein are released by iterative incubation in a buffered organic solvent system ( 2 ). To increase Digestion efficiency and sequence coverage proteins are reduced ( 3 ) and alkylated ( 4 ). Proteins are subsequently digested with proteolytic enzymes ( scissors symbols ), typically trypsin ( 5 ). Trypsin cleaves at the amino acid residues arginine ( R ) and lysine ( K ). The resulting peptides ( A , B , and C ) are extracted from the polyacrylamide matrix ( 6 ). The peptide solution can be further purified for analysis by mass spectrometry (Section “ Concentration and desalting of peptides ”)
Matthias Mann - One of the best experts on this subject based on the ideXlab platform.
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In-Gel Digestion for mass spectrometric characterization of proteins and proteomes
Nature Protocols, 2007Co-Authors: Andrej Shevchenko, Henrik Tomas, Jan Havliš, Jesper V. Olsen, Matthias MannAbstract:In-Gel Digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the In-Gel Digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
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Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations
Analytical Chemistry, 1999Co-Authors: Gitte Neubauer, Matthias MannAbstract:: Precursor ion scans have proven to be extremely useful for the characterization of unseparated peptide mixtures. In conjunction with the nanoelectrospray source, precursor ion scans provide a sensitive tool for the detection of posttranslationally modified peptides and have been used to determine phosphorylation sites of proteins digested in solution. In this report, we extend our previous work to the determination of protein phosphorylation sites of gel-isolated proteins. The In-Gel Digestion procedure developed in our laboratory for protein microsequencing was found to be suitable for phosphorylation mapping as well. The risk of losing hydrophilic peptides in the desalting step was decreased by using column packing material designed for the purification of oligonucleotides and by adjusting the pH conditions to the needs of phosphopeptide analysis. With this method, the tryptic phosphopeptides of beta-casein were detected after In-Gel Digestion at a sensitivity of 250 fmol of protein applied to the gel. The phosphorylation sites of two other proteins, Src-delta U and Op18, have similarly been mapped. Subpicomole to low-picomole amounts of protein starting material are needed in general, although we and others have reported attomole sensitivity for the detection of model phosphopeptides using precursor ion scans. This indicates that the success in determining phosphorylation sites depends crucially on the Digestion, extraction, and detection efficiency for individual phosphopeptides.
