1995
DOI: 10.1002/elps.1150160171
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Abstract: Amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two-dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS-PRO… Show more

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Cited by 124 publications
(32 citation statements)
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“…Two-dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)-Cell pellets were resuspended in lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1% sulfobetaine-3-10, 1% amidosulfobetaine-14, 2 mM tributylphosphine, 65 mM dithiothreitol, 1% carrier ampholyte (pH range 3-10), 1% carrier ampholyte (pH range 4 -6), 0.01% bromphenol blue) to a final concentration of 1 mg/ml as determined by amino acid analysis (30). Cells were lysed by pulse sonication twice for 10 s on ice.…”
Section: Methodsmentioning
confidence: 99%
“…Two-dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)-Cell pellets were resuspended in lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1% sulfobetaine-3-10, 1% amidosulfobetaine-14, 2 mM tributylphosphine, 65 mM dithiothreitol, 1% carrier ampholyte (pH range 3-10), 1% carrier ampholyte (pH range 4 -6), 0.01% bromphenol blue) to a final concentration of 1 mg/ml as determined by amino acid analysis (30). Cells were lysed by pulse sonication twice for 10 s on ice.…”
Section: Methodsmentioning
confidence: 99%
“…Samples for matrix-assisted laser desorption ionization-time of fl ight (MALDI-TOF) analysis were excised from the gels. In-gel digestion of the individual protein spots was done using 12.5 μg/ml trypsin (Wako Pure Chemical Industry, Osaka, Japan) as described previously (3,7,14,24). The peptide solutions were vacuum-concentrated until the volume was decreased to 10 μl, and desalted using ZipTip™ C18 column (Millipore, Bedford, MA, USA) according to the manufacturer's Fig.…”
Section: Identifi Cation Of Protein Spots On Two Dimensional Gelsmentioning
confidence: 99%
“…Proteomic technologies using two dimensional gel electrophoresis and mass spectrometry provide the tool to discover and identify novel genes or proteins (3,7,14,24).…”
mentioning
confidence: 99%
“…Such techniques would be incomplete without database algorithms used to analyse the output data for suitable database matches. These programs have been used as tools for single-species (homologous) protein identification and some cross-species (heterologous) characterisations [9][10]231. In order to better understand the confidence gained from matches obtained using these programs and their corresponding analytical techniques, proteins from three fully sequenced microbial genomes (Haemophilus influenzae [24], Mycoplasma genitalium [25], and Methanococcus jannaschii [26]) were used to assess the ability of search programs to successfully find matches across species boundaries.…”
Section: Introductionmentioning
confidence: 99%
“…These sequences were analysed in the SWISS-PROT database using the program FASTA [32]. N-Terminal protein sequences were searched using a modified FASTA, designed to examine only the first twenty residues of SWISS-PROT entries, as detailed previously [9].…”
Section: Introductionmentioning
confidence: 99%