Abstract
We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI-TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.
MeSH terms
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Antibodies, Phospho-Specific / analysis
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Blotting, Western / methods
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Carcinoma, Squamous Cell
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Cell Extracts / chemistry*
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Cell Line, Tumor / chemistry
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Cell Line, Tumor / drug effects
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Cell Membrane / chemistry
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Electrophoresis, Gel, Two-Dimensional / methods*
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Epidermal Growth Factor / pharmacology
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Humans
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Molecular Weight
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Peptide Mapping*
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Phosphoproteins / analysis*
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Phosphoproteins / immunology
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Phosphotyrosine / analysis*
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Phosphotyrosine / immunology
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Proteins / isolation & purification*
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
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Tandem Mass Spectrometry / methods
Substances
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Antibodies, Phospho-Specific
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Cell Extracts
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Phosphoproteins
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Proteins
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Phosphotyrosine
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Epidermal Growth Factor