The Experts below are selected from a list of 49881 Experts worldwide ranked by ideXlab platform
George N Phillips - One of the best experts on this subject based on the ideXlab platform.
-
crystal structure of human protein n terminal glutamine amidohydrolase an initial component of the n end rule pathway
PLOS ONE, 2014Co-Authors: Mi Seul Park, E Bitto, Kyung Rok Kim, Craig A Bingman, Mitchell D Miller, Hyunjung Kim, Byung Woo Han, George N PhillipsAbstract:The N-end rule states that half-life of protein is determined by their N-Terminal amino acid Residue. N-Terminal glutamine amidohydrolase (Ntaq) converts N-Terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 A resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-Terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-Terminal peptides of substrate proteins. Upon binding of a substrate with N-Terminal glutamine, active site catalytic triad mediates the deamination of the N-Terminal Residue to glutamate by a mechanism analogous to that of cysteine proteases.
Mikhail V. Gorshkov - One of the best experts on this subject based on the ideXlab platform.
-
combination of edman degradation of peptides with liquid chromatography mass spectrometry workflow for peptide identification in bottom up proteomics
Rapid Communications in Mass Spectrometry, 2013Co-Authors: Anna A. Lobas, Anatoly N. Verenchikov, Anton Goloborodko, Lev I. Levitsky, Mikhail V. GorshkovAbstract:RATIONALE High-throughput methods of proteomics are essential for identification of proteins in a cell or tissue under certain conditions. Most of these methods require tandem mass spectrometry (MS/MS). A multidimensional approach including predictive chromatography and partial chemical degradation could be a valuable alternative and/or addition to MS/MS. METHODS In the proposed strategy peptides are identified in a three-dimensional (3D) search space consisting of retention time (RT), mass, and reduced mass after one-step partial Edman degradation. The strategy was evaluated in silico for two databases: baker's yeast and human proteins. Rates of unambiguous identifications were estimated for mass accuracies from 0.001 to 0.05 Da and RT prediction accuracies from 0.1 to 5 min. Rates of Edman reactions were measured for test peptides. RESULTS A 3D description of proteolytic peptides allowing unambiguous identification without employing MS/MS of up to 95% and 80% of tryptic peptides from the yeast and human proteomes, respectively, was considered. Further extension of the search space to a four-dimensional one by incorporating the second N-Terminal amino acid Residue as the fourth dimension was also considered and was shown to result in up to 90% of human peptides being identified unambiguously. CONCLUSIONS The proposed 3D search space can be a useful alternative to MS/MS-based peptide identification approach. Experimental implementations of the proposed method within the on-line liquid chromatography/mass spectrometry (LC/MS) and off-line matrix-assisted laser desorption/ionization (MALDI) workflows are in progress. Copyright © 2012 John Wiley & Sons, Ltd.
Zhonghuai Xiang - One of the best experts on this subject based on the ideXlab platform.
-
proteomic analysis of spore wall proteins and identification of two spore wall proteins from nosema bombycis microsporidia
Proteomics, 2008Co-Authors: Guoqing Pan, Xiaohui Tan, Zeyang Zhou, Zhonghuai XiangAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-Terminal amino acid Residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.
Zeyang Zhou - One of the best experts on this subject based on the ideXlab platform.
-
proteomic analysis of spore wall proteins and identification of two spore wall proteins from nosema bombycis microsporidia
Proteomics, 2008Co-Authors: Guoqing Pan, Xiaohui Tan, Zeyang Zhou, Zhonghuai XiangAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-Terminal amino acid Residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.
Anton Goloborodko - One of the best experts on this subject based on the ideXlab platform.
-
combination of edman degradation of peptides with liquid chromatography mass spectrometry workflow for peptide identification in bottom up proteomics
Rapid Communications in Mass Spectrometry, 2013Co-Authors: Anna A. Lobas, Anatoly N. Verenchikov, Anton Goloborodko, Lev I. Levitsky, Mikhail V. GorshkovAbstract:RATIONALE High-throughput methods of proteomics are essential for identification of proteins in a cell or tissue under certain conditions. Most of these methods require tandem mass spectrometry (MS/MS). A multidimensional approach including predictive chromatography and partial chemical degradation could be a valuable alternative and/or addition to MS/MS. METHODS In the proposed strategy peptides are identified in a three-dimensional (3D) search space consisting of retention time (RT), mass, and reduced mass after one-step partial Edman degradation. The strategy was evaluated in silico for two databases: baker's yeast and human proteins. Rates of unambiguous identifications were estimated for mass accuracies from 0.001 to 0.05 Da and RT prediction accuracies from 0.1 to 5 min. Rates of Edman reactions were measured for test peptides. RESULTS A 3D description of proteolytic peptides allowing unambiguous identification without employing MS/MS of up to 95% and 80% of tryptic peptides from the yeast and human proteomes, respectively, was considered. Further extension of the search space to a four-dimensional one by incorporating the second N-Terminal amino acid Residue as the fourth dimension was also considered and was shown to result in up to 90% of human peptides being identified unambiguously. CONCLUSIONS The proposed 3D search space can be a useful alternative to MS/MS-based peptide identification approach. Experimental implementations of the proposed method within the on-line liquid chromatography/mass spectrometry (LC/MS) and off-line matrix-assisted laser desorption/ionization (MALDI) workflows are in progress. Copyright © 2012 John Wiley & Sons, Ltd.
