Electrospray Ionization Mass Spectrometry on Hydrophobic Peptides Electroeluted from Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Application to the Topology of the Sarcoplasmic Reticulum Ca2+ATPase

We describe a method to prepare proteins and pep-tides in a state suitable for exact determination of molecular mass by electrospray ionization mass spectrometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution. The utility of the procedure, in conjunction with N-terminal sequencing, in defining the C-terminal end of the peptide fragments produced by proteolysis of sarcoplasmic reticulum Ca2+ATPase with V8 is demonstrated. The application of mass spectrometry aids significantly the use of proteolytic enzymes for topological studies of membrane proteins, and SDS-PAGE is preferable to reverse-phase HPLC for separation of membraneous, hydrophobic peptides and proteins.