Md. Bauer et al., Sequencing of gel-isolated proteins using microblotter capillary liquid chromatography-electrospray mass spectrometry, J PROTEIN C, 18(3), 1999, pp. 337-347
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.