A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage.
Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82-99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.
Distler
Anne M
AM
Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
Kerner
Janos
J
Peterman
Scott M
SM
Hoppel
Charles L
CL
D000595:Amino Acid Sequence; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000032:analysis / Q000235:genetics; D003066:Coenzyme A Ligases / Q000032:analysis / Q000235:genetics; D019286:Endopeptidase K; D008297:Male; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D008930:Mitochondria, Liver / Q000737:chemistry*; D024101:Mitochondrial Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin; D050994:Voltage-Dependent Anion Channels / Q000032:analysis / Q000235:genetics
D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D050994:Voltage-Dependent Anion Channels; D002334:Carnitine O-Palmitoyltransferase; D014357:Trypsin; D019286:Endopeptidase K; D003066:Coenzyme A Ligases