Visualization of proteins by modification of lysines, cysteines, and phosphorylated serines facilitates sample preparation for microsequencing.
A procedure for visualization and sensitive detection of protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent sample preparation for sequence analysis is described. This procedure utilizes either fluorescent or visible tags for certain amino acids in protein molecules, e.g., lysines modified with dansyl/dabsyl chloride and cystines/cysteines or phosphorylated serines modified with iodoacetamidofluorescein (I-15) after proper sample pretreatments. Modifications are performed prior to SDS-PAGE, eliminating the need for fixing, staining, and destaining as required for the conventional procedures. After electrophoresis, the fluorescent or visible bands are excised from the gel, homogenized in microcentrifuge tubes, and soaked in an appropriate buffer to release the separated proteins into solution. Enzymatic digestion can then be carried out in solution for better efficiency of digestion and recovery. The subsequent HPLC mapping and collection of protein digests are performed on PE Applied Biosystems Model 173A MicroBlotter. The separated peptides containing tagged amino acids are visible on the PVDF membrane and can be excised for direct sequence analysis. This approach has been employed for selectively isolating the lysine, cysteine, or phosphorylated serine containing peptides using model proteins. The sequencing results of the peptides generated from premodified proteins demonstrate that this approach facilitates sample preparation for microsequence analysis at low picomole level. Overall recoveries of 20-30% by sequencing initial yields have been achieved using our model proteins.
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PE Applied Biosystems, 850 Lincoln Center Drive, Foster City, California 94404, USA. Hsik@Perkin-Elmer.com
O'Neill
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Dupont
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Yuan
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D017421:Sequence Analysis / Q000379:methods*; D012694:Serine / Q000737:chemistry; D014357:Trypsin
D011506:Proteins; D012694:Serine; D014357:Trypsin; D008239:Lysine; D003545:Cysteine