title: Extending the dynamic range of label-free mass spectrometric quantification of affinity purifications.
abstract: Affinity purification (AP) of protein complexes combined with LC-MS/MS analysis is the current method of choice for identification of protein-protein interactions. Their interpretation with respect to significance, specificity, and selectivity requires quantification methods coping with enrichment factors of more than 1000-fold, variable amounts of total protein, and low abundant, unlabeled samples. We used standardized samples (0.1-1000 fmol) measured on high resolution hybrid linear ion trap instruments (LTQ-FT/Orbitrap) to characterize and improve linearity and dynamic range of label-free approaches. Quantification based on spectral counts was limited by saturation and ion suppression effects with samples exceeding 100 ng of protein, depending on the instrument setup. In contrast, signal intensities of peptides (peak volumes) selected by a novel correlation-based method (TopCorr-PV) were linear over at least 4 orders of magnitude and allowed for accurate relative quantification of standard proteins spiked into a complex protein background. Application of this procedure to APs of the voltage-gated potassium channel Kv1.1 as a model membrane protein complex unambiguously identified the whole set of known interaction partners together with novel candidates. In addition to discriminating these proteins from background, we could determine efficiency, cross-reactivities, and selection biases of the used purification antibodies. The enhanced dynamic range of the developed quantification procedure appears well suited for sensitive identification of specific protein-protein interactions, detection of antibody-related artifacts, and optimization of AP conditions.
authors: Institute of Physiology, University of Freiburg, 79104 Freiburg, Germany.
authors: Wolfgang
authors: W
authors: Bildl
authors: Alexander
authors: A
authors: Haupt
authors: Catrin S
authors: CS
authors: Müller
authors: Martin L
authors: ML
authors: Biniossek
authors: Jörg Oliver
authors: JO
authors: Thumfart
authors: Björn
authors: B
authors: Hüber
authors: Bernd
authors: B
authors: Fakler
authors: Uwe
authors: U
authors: Schulte
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D005583:Fourier Analysis; D051662:Kv1.1 Potassium Channel / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D040901:Proteomics; D051381:Rats; D053719:Tandem Mass Spectrometry
chemical_list: D051662:Kv1.1 Potassium Channel