Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry.
Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.
NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, Reutlingen, Germany.
Frederik
F
Weiß
Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany.
Anke
A
Schnabel
NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, Reutlingen, Germany.
Hannes
H
Planatscher
NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, Reutlingen, Germany.
Bart H J
BH
van den Berg
Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany.
Bettina
B
Serschnitzki
Department of Traumatology, Eberhard Karls Universität Tübingen, Tuebingen, Germany.
Andreas K
AK
Nuessler
Department of Surgery, Ludwig-Maximilians-University, Munich, Germany.
Wolfgang E
WE
Thasler
Department of Pediatrics and Juvenile Medicine, Regensburg University Hospital, Regensburg, Germany.
Thomas S
TS
Weiss
Center Systems Biology, University of Stuttgart, Stuttgart, Germany.
Matthias
M
Reuss
University of Applied Sciences, Albstadt Sigmaringen, Germany.
Dieter
D
Stoll
NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, Reutlingen, Germany.
Markus F
MF
Templin
NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, Reutlingen, Germany.
Thomas O
TO
Joos
Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany.
Katrin
K
Marcus
NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, Reutlingen, Germany.
Oliver
O
Poetz
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000915:Antibody Affinity; D001189:Aryl Hydrocarbon Hydroxylases / Q000276:immunology / Q000378:metabolism; D000069059:Atorvastatin / Q000493:pharmacokinetics; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D051544:Cytochrome P-450 CYP3A / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism*; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000493:pharmacokinetics; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000276:immunology / Q000378:metabolism*; D017035:Pravastatin / Q000493:pharmacokinetics; D061251:Primary Cell Culture; D017386:Sequence Homology, Amino Acid
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000906:Antibodies; D000939:Epitopes; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D010455:Peptides; D000069059:Atorvastatin; D001189:Aryl Hydrocarbon Hydroxylases; C103123:CYP3A7 protein, human; D051544:Cytochrome P-450 CYP3A; D017035:Pravastatin