Direct mass spectrometric identification of ABCB1 (P-glycoprotein/MDR1) from the apical membrane fraction of human placenta using fourier transform ion cyclotron mass spectrometry.
Identification and quantification of proteins from human tissue by electrophoresis and subsequent mass spectrometry (MS) has been mainly restricted to high abundance, non-membrane-bound molecules. Pharmacologically relevant structures such as cytochrome P450 enzyme, drug transporters and receptors were not accessible by this technology. We developed a method to identify an integral membrane-bound protein (ABCB1) from human placenta using Fourier transform ion cyclotron (FTICR)-MS. Apical and basal membrane fractions were enriched from term human placenta using differential centrifugation. Following SDS-page these fractions were cleaved with trypsin, separated by nano-HPLC and subjected to FTICR-MS. We identified a total of 70 and 89 proteins in the apical and basal membranes, respectively. Among these proteins, and restricted to the apical membrane, was the transport protein ABCB1, with 10 peptides identified by MS, covering a total of 10% of the entire protein. The study describes a method suitable for direct monitoring of membrane-bound proteins from human tissue using FTICR-MS.
Meyer zu Schwabedissen
Henriette E
HE
Department of Pharmacology, Peter-Holtz Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Dreisbach
Annette
A
Hammer
Elke
E
Fusch
Christoph
C
Hecker
Michael
M
Völker
Uwe
U
Kroemer
Heyo K
HK
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D017357:Cyclotrons; D005260:Female; D019450:Genes, MDR; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D027361:Organic Anion Transporters / Q000032:analysis*; D010920:Placenta / Q000737:chemistry*; D011247:Pregnancy; D017550:Spectroscopy, Fourier Transform Infrared
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008565:Membrane Proteins; D027361:Organic Anion Transporters