Quantitative interaction proteomics using mass spectrometry.
We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.
Wepf
Alexander
A
Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, Eidgenössische Technische Hochschule Zürich, Wolfgang Pauli-Strasse 16, Zurich, Switzerland.
Glatter
Timo
T
Schmidt
Alexander
A
Aebersold
Ruedi
R
Gstaiger
Matthias
M
D000345:Affinity Labels / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D019319:Okadaic Acid / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014162:Transfection
D000345:Affinity Labels; D046912:Multiprotein Complexes; C517191:PPP2R1A protein, human; D010455:Peptides; D019319:Okadaic Acid; D002265:Carboxylic Ester Hydrolases; C119357:protein phosphatase methylesterase-1; C517189:PPP2CA protein, human; C517190:PPP2CB protein, human; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C080406:protein phosphatase 4