The phosphorylation site and desmethionyl N-terminus of Drosophila phosrestin I in vivo determined by mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis

1997 ◽  
Vol 3 (1) ◽  
pp. 367 ◽  
Author(s):  
Tomoya Kinumi ◽  
Sara Tobin ◽  
Hiroyuki Matsumoto ◽  
Kenneth Jackson ◽  
Mamoru Ohashi
Keyword(s):  
Gel Electrophoresis ◽  
Phosphorylation Site ◽  
Mass Spectrometric ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
N Terminus

scholarly journals TDP-l-Megosamine Biosynthesis Pathway Elucidation and Megalomicin A Production in Escherichia coli

2010 ◽  
Vol 76 (12) ◽  
pp. 3869-3877 ◽  
Author(s):  
Mariana Useglio ◽  
Salvador Peirú ◽  
Eduardo Rodríguez ◽  
Guillermo R. Labadie ◽  
John R. Carney ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Mass Spectrometric ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Biosynthesis Pathway ◽  
Biosynthetic Genes

ABSTRACT In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.

scholarly journals Altered Protein Expression of Streptococcus oralis Cultured at Low pH Revealed by Two-Dimensional Gel Electrophoresis

2001 ◽  
Vol 67 (8) ◽  
pp. 3396-3405 ◽  
Author(s):  
Joanna C. Wilkins ◽  
Karen A. Homer ◽  
David Beighton
Keyword(s):  
Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Low Ph ◽  
Ionization Mass ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Altered Expression ◽  
Streptococcus Oralis ◽  
Peptide Mass

ABSTRACT Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database forStreptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.

In vivo distribution of proteins within a single cell.

1982 ◽  
Vol 28 (4) ◽  
pp. 1011-1014 ◽  
Author(s):  
C F Austerberry ◽  
P L Paine
Keyword(s):  
Xenopus Laevis ◽  
Single Cell ◽  
Gel Electrophoresis ◽  
Living Cell ◽  
Experimental System ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
In Vivo Distribution ◽  
Nucleocytoplasmic Distribution

Abstract Using the oocyte of Xenopus laevis, we present an experimental system, involving two-dimensional gel electrophoresis, for measuring unambiguously the nucleocytoplasmic distribution of proteins within a living cell.

scholarly journals Initial Proteome Analysis of Model Microorganism Haemophilus influenzae Strain Rd KW20

2003 ◽  
Vol 185 (15) ◽  
pp. 4593-4602 ◽  
Author(s):  
Eugene Kolker ◽  
Samuel Purvine ◽  
Michael Y. Galperin ◽  
Serg Stolyar ◽  
David R. Goodlett ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Protein Identification ◽  
Proteome Analysis ◽  
Tandem Mass ◽  
Two Dimensional ◽  
High Confidence ◽  
Two Dimensional Gel Electrophoresis

ABSTRACT The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism of H. influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) of H. influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.

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