Sequencing of gel-isolated proteins using microblotter capillary liquid chromatography-electrospray mass spectrometry

Enzymatic digests of proteins isolated by sodium dodecyl sulfate-polyacryla mide gel electrophoresis (SDS-PAGE) were separated by capillary high-perfor mance liquid chromatography (HPLC). The column eluate was split to an elect rospray mass spectrometer on one side and to both a UV detector and a micro blotter 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 spec trometric analysis and a prepared PVDF membrane for subsequent Edman sequen cing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most imp ortant peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated sys tem for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for prot ein characterization. Proteins loaded onto SDS-PAGE at low picomole levels can be analyzed by the new integrated system.