The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Peter Roepstorff - One of the best experts on this subject based on the ideXlab platform.
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chemical cross linking with thiol cleavable reagents combined with differential mass spectrometric Peptide Mapping a novel approach to assess intermolecular protein contacts
Protein Science, 2000Co-Authors: Keiryn L Bennett, Martin Kussmann, Per Bjork, Magdalena Godzwon, Marie Mikkelsen, Poul Sorensen, Peter RoepstorffAbstract:The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS Peptide Mapping. Comparison of the Peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS Peptide Mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS Peptide Mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.
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matrix assisted laser desorption ionization mass spectrometric Peptide Mapping of the neural cell adhesion protein neurolin purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis or acidic precipitation
Journal of Mass Spectrometry, 1997Co-Authors: Martin Kussmann, Ute Lassing, Claudia Sturmer, Michael Przybylski, Peter RoepstorffAbstract:Neurolin is a cell surface protein involved in the neural regeneration and neogenesis of the central nervous system of goldfish. Its theoretical molecular mass, based on the amino acid sequence translated from the cDNA, is 58 kDa, but in SDS-PAGE it shows an apparent MW of 86 kDa. Neurolin is stated to be a glycoprotein and it contains five potential N- and 96 potential O-glycosylation sites. The complete characterization of the primary structure and initial investigations on the postulated glycosylation of neurolin, immunopurified from goldfish brains, are described. The protein was either digested in situ in the sodium dodecyl sulfate polyacrylamide gel matrix or digested after trichloroacetic acid precipitation. Trypsin and endoprotease Glu-C were used as proteases and matrix-assisted laser desorption/ionization mass spectrometry was applied for direct Peptide Mapping analysis of the proteolytic mixtures. Various sample preparation techniques were performed and the mass spectra were recorded in both positive- and negative-ion modes.
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identification of proteins in polyacrylamide gels by mass spectrometric Peptide Mapping combined with database search
Journal of Mass Spectrometry, 1994Co-Authors: Ejvind Mortz, Matthias Mann, Ole Vorm, Peter RoepstorffAbstract:Mass spectrometric Peptide Mapping of proteins separated by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis has been investigated. The best results are obtained after blotting of the proteins onto polyvinylidene difluoride membranes followed by enzymatic digestion of the protein on the membrane. The Peptide maps were investigated in terms of completeness and applicability for protein identification using a previously developed database search program as well as for the possibility for full characterization of covalent modifications in the proteins. The most complete Peptide maps were obtained when the proteins were reduced and alkylated on the membrane prior to enzymatic digestion followed by separation of the resulting mixture by high performance liquid chromatography prior to mass spectrometric analysis. Such Peptide maps cover up to 98% of the sequence and consequently may allow complete characterization of post-translational modifications in proteins for which the amino acid sequence is known. The fastest and most sensitive procedure to obtain Peptide maps sufficient for protein identification was direct analysis of the extracted Peptide mixture by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The use of external and internal calibration of MALDI spectra for database searches is evaluated as well as the possibility of including a post-calibration routine within the search program.
Michael Przybylski - One of the best experts on this subject based on the ideXlab platform.
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Mass Spectrometric Peptide Mapping Analysis and Structural Characterization of
2013Co-Authors: Dihydrodiol Dehydrogenase Lsoenzymes, Christine Gauss, Jochen Klein, Karin T Post, T Detlev, Klaus Schneider, Helmut Thomas, T Franz, Michael PrzybylskiAbstract:The direct molecular weight determination and structural analysis of polyPeptides and Peptide mixtures have become amenable by the recent development of fast atom bombardment (FABMS) and 252Cf-plasma desorption (PDMS) mass spectrometry. FABMS and PDMS Peptide Mapping, i.e., the direct analysis of Peptide mixtures resulting from proteolytic digestion, have been developed as powerful methods for the structural characterization of epoxide-metabolizing isoenzymes. The major advantage of this approach is provided by the selectivity of the endoproteolytic cleavage, combined with the specific and accurate molecular weight determination of complex digest mixtures containing Peptides up to several thousand daltons in size. Furthermore, the mass spectrometric Peptide Mapping analysis can be combined with a range of protein-chemical modification reactions and with sequential degradation such as by carboxypeptidases. Both FABMS and PDMS Peptide Mapping have already been successfully applied to the structural differentiation of glutathione transferase and epoxide hydrolase isoenzymes in cases where reference sequence data for at least one isoenzyme form was available. In the application described here, for a series of dihydrodiol dehydrogenase (DDH) isoenzymes with hitherto undetermined primary structures, a direct correlation between the structural differentiation from Peptide Mapping data and differences in their substrate specificities could be demonstrated. The mass spectrometric Peptide Mapping analysis of isoenzymes proved to be an efficient basis for the elucidation of the structure of one major DDH isoenzyme form; partial sequence data for this protein are reported
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matrix assisted laser desorption ionization mass spectrometric Peptide Mapping of the neural cell adhesion protein neurolin purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis or acidic precipitation
Journal of Mass Spectrometry, 1997Co-Authors: Martin Kussmann, Ute Lassing, Claudia Sturmer, Michael Przybylski, Peter RoepstorffAbstract:Neurolin is a cell surface protein involved in the neural regeneration and neogenesis of the central nervous system of goldfish. Its theoretical molecular mass, based on the amino acid sequence translated from the cDNA, is 58 kDa, but in SDS-PAGE it shows an apparent MW of 86 kDa. Neurolin is stated to be a glycoprotein and it contains five potential N- and 96 potential O-glycosylation sites. The complete characterization of the primary structure and initial investigations on the postulated glycosylation of neurolin, immunopurified from goldfish brains, are described. The protein was either digested in situ in the sodium dodecyl sulfate polyacrylamide gel matrix or digested after trichloroacetic acid precipitation. Trypsin and endoprotease Glu-C were used as proteases and matrix-assisted laser desorption/ionization mass spectrometry was applied for direct Peptide Mapping analysis of the proteolytic mixtures. Various sample preparation techniques were performed and the mass spectra were recorded in both positive- and negative-ion modes.
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protein surface topology probing by selective chemical modification and mass spectrometric Peptide Mapping
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Detlev Suckau, Marianna Mak, Michael PrzybylskiAbstract:Abstract Aminoacetylation of lysine residues and the modification of arginine by 1,2-cyclohexanedione to N7,N8-(dihydroxy-1,2-cyclohexylidene)arginine were used for probing the surface topology of hen-eggwhite lysozyme as a model protein. The molecular identification of lysine and arginine modification sites was provided by molecular weight determinations of modified and unmodified tryptic Peptide mixtures (Peptide Mapping) using 252Cf plasma desorption mass spectrometry. At conditions of limited chemical modification, mass-spectrometric Peptide-Mapping analyses of lysozyme derivatives enabled the direct assignment of relative reactivities of lysine and arginine residues at different reaction times and reagent concentrations. The relative reactivities of lysine residues showed a direct correlation with their surface accessibilities from x-ray structure data. For the reaction with 1,2-cyclohexanedione, a selective modification at Arg-5, -125, -112, and -73 was identified, and an inverse correlation of relative reactivities with the surface accessibility ratios of the N7- and the N8-guanidino functions was obtained. By examination of the x-ray structural data of lysozyme, this selective modification was attributed to intramolecular catalysis because of the presence of neighboring proton acceptor groups, such as the Asp-119 carboxylate group for Arg-125 and the Trp-123 and Arg-125 carbonyl groups for Arg-5.
Brian T. Chait - One of the best experts on this subject based on the ideXlab platform.
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ProFound: An expert system for protein identification using mass spectrometric Peptide Mapping information
Analytical Chemistry, 2000Co-Authors: Wenzhu Zhang, Brian T. ChaitAbstract:We describe the protein search engine "ProFound", which employs a Bayesian algorithm to identify proteins from protein databases using mass spectrometric Peptide Mapping data. The algorithm ranks protein candidates by taking into account individual properties of each protein in the database as well as other information relevant to the Peptide Mapping experiment. The program consistently identifies the correct protein(s) even when the data quality is relatively low or when the sample consists of a simple mixture of proteins. Illustrative examples of protein identifications are provided.
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modification of cysteine residues by alkylation a tool in Peptide Mapping and protein identification
Analytical Chemistry, 1998Co-Authors: Salvatore Sechi, Brian T. ChaitAbstract:Although mass spectrometric Peptide Mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of Peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Peptide Mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide...
Gang Chen - One of the best experts on this subject based on the ideXlab platform.
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infrared assisted proteolysis using trypsin immobilized silica microspheres for Peptide Mapping
Proteomics, 2009Co-Authors: Huimin Bao, Luyan Zhang, Ting Lui, Gang ChenAbstract:A novel proteolysis approach was developed by using infrared (IR) radiation and trypsin-immobilized silica microspheres. Protein solutions containing trypsin-immobilized microspheres in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 degrees C. The feasibility and performance of the present proteolysis approach were demonstrated by the digestion of BSA and cytochrome c (Cyt-c) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 54% (BSA) and 83% (Cyt-c) that were better than those obtained by conventional in-solution tryptic digestion. The suitability of the new digestion approach to complex proteins was demonstrated by digesting human serum. The present proteolysis strategy is simple and efficient and will find a wide range of application in protein identification.
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accelerated proteolysis in alternating electric fields for Peptide Mapping
Rapid Communications in Mass Spectrometry, 2008Co-Authors: Sheng Wang, Luyan Zhang, Pengyuan Yang, Ting Liu, Huimin Bao, Gang ChenAbstract:Sinusoidal alternating voltages (typically 5 V) were employed to enhance the efficiency of proteolysis for Peptide Mapping in this work. Protein solutions containing trypsin were allowed to digest with the assistance of alternating electric fields (AEFs) between a pair of platinum wire electrodes in Eppendorf tubes. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of several standard proteins. It was demonstrated that AEFs significantly accelerated in-solution proteolysis and the digestion time was substantially reduced to 5 min. The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with sequence coverages that were comparable to those obtained by using conventional 12-h in-solution proteolysis. The suitability of AEF-assisted proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). The present proteolysis strategy is simple and efficient and will find a wide range of applications in protein identification.
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efficient chymotryptic proteolysis enhanced by infrared radiation for Peptide Mapping
Journal of Proteome Research, 2008Co-Authors: Sheng Wang, Luyan Zhang, Ting Liu, Gang ChenAbstract:Infrared (IR) radiation was employed to enhance the efficiency of chymotryptic proteolysis for Peptide Mapping in this work. Protein solutions containing chymotrypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 °C. BSA and cytochrome c (Cyt-c) were digested by IR-assisted chymotryptic proteolysis to demonstrate the feasibility and performance of the novel digestion approach and the digestion time was significantly reduced to 5 min. The obtained digests were further identified by MALDI-TOF MS with the sequence coverages that were comparable to those obtained by using conventional in-solution digestion. The suitability of IR-assisted chymotryptic proteolysis to complex proteins was demonstrated by digesting human serum. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.
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infrared assisted tryptic proteolysis for Peptide Mapping
Proteomics, 2008Co-Authors: Sheng Wang, Luyan Zhang, Pengyuan Yang, Gang ChenAbstract:In this report, infrared (IR) radiation was employed to enhance the efficiency of tryptic proteolysis for Peptide Mapping. Protein solutions containing trypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 degrees C. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of BSA and myoglobin (MYO) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 69% (BSA) and 90% (MYO) that were much better than those obtained by conventional in-solution tryptic digestion. The present IR-assisted proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.
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infrared assisted on plate proteolysis for maldi tof ms Peptide Mapping
Analytical Chemistry, 2008Co-Authors: Sheng Wang, Luyan Zhang, Pengyuan Yang, Huimin Bao, Gang ChenAbstract:In this report, infrared (IR)-assisted on-plate proteolysis has been developed for rapid Peptide Mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of matrix-assisted laser desorption/ionization (MALDI) plates under IR radiation. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c). It was demonstrated that IR radiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI time-of-flight mass spectrometry with sequence coverages of 55 (BSA) and 75% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.
Martin Kussmann - One of the best experts on this subject based on the ideXlab platform.
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chemical cross linking with thiol cleavable reagents combined with differential mass spectrometric Peptide Mapping a novel approach to assess intermolecular protein contacts
Protein Science, 2000Co-Authors: Keiryn L Bennett, Martin Kussmann, Per Bjork, Magdalena Godzwon, Marie Mikkelsen, Poul Sorensen, Peter RoepstorffAbstract:The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS Peptide Mapping. Comparison of the Peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS Peptide Mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS Peptide Mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.
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matrix assisted laser desorption ionization mass spectrometric Peptide Mapping of the neural cell adhesion protein neurolin purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis or acidic precipitation
Journal of Mass Spectrometry, 1997Co-Authors: Martin Kussmann, Ute Lassing, Claudia Sturmer, Michael Przybylski, Peter RoepstorffAbstract:Neurolin is a cell surface protein involved in the neural regeneration and neogenesis of the central nervous system of goldfish. Its theoretical molecular mass, based on the amino acid sequence translated from the cDNA, is 58 kDa, but in SDS-PAGE it shows an apparent MW of 86 kDa. Neurolin is stated to be a glycoprotein and it contains five potential N- and 96 potential O-glycosylation sites. The complete characterization of the primary structure and initial investigations on the postulated glycosylation of neurolin, immunopurified from goldfish brains, are described. The protein was either digested in situ in the sodium dodecyl sulfate polyacrylamide gel matrix or digested after trichloroacetic acid precipitation. Trypsin and endoprotease Glu-C were used as proteases and matrix-assisted laser desorption/ionization mass spectrometry was applied for direct Peptide Mapping analysis of the proteolytic mixtures. Various sample preparation techniques were performed and the mass spectra were recorded in both positive- and negative-ion modes.