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 peptides 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.
le Maire
M
M
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Deschamps
S
S
Møller
J V
JV
Le Caer
J P
JP
Rossier
J
J
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000737:chemistry; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012697:Serine Endopeptidases; D012967:Sodium Dodecyl Sulfate
D010446:Peptide Fragments; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D012967:Sodium Dodecyl Sulfate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D000252:Calcium-Transporting ATPases