Sequencing of gel-isolated proteins using microblotter capillary liquid chromatography-electrospray mass spectrometry.
Enzymatic digests of proteins isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were separated by capillary high-performance liquid chromatography (HPLC). The column eluate was split to an electrospray mass spectrometer on one side and to both a UV detector and a microblotter on the other side. Using the microblotter, the peptides eluted from the column were collected directly onto a polyvinylidene difluoride (PVDF) membrane for Edman sequencing. Thus, a peptide mass map from the mass spectrometric analysis and a prepared PVDF membrane for subsequent Edman sequencing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most important peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated system for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for protein characterization. Proteins loaded onto SDS-PAGE at low picomole levels can be analyzed by the new integrated system.
The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45253-8707, USA.
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Bauer
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Sun
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Wang
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003574:Cytochrome c Group / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D006602:Hexosyltransferases; D007782:Lactoglobulins / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013381:Subtilisins / Q000737:chemistry; D013997:Time Factors
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003574:Cytochrome c Group; D007782:Lactoglobulins; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D013381:Subtilisins