Intelligent Mixing of Proteomes for Elimination of False Positives in Affinity Purification-Mass Spectrometry

Abstract

Protein complexes are essential in all organizational and functional aspects of the cell. Different strategies currently exist for analyzing such protein complexes by mass spectrometry, including affinity purification (AP-MS) and proximal labeling-based strategies. However, the high sensitivity of current mass spectrometers typically results in extensive protein lists mainly consisting of nonspecifically copurified proteins. Finding the true positive interactors in these lists remains highly challenging. Here, we report a powerful design based on differential labeling with stable isotopes combined with nonequal mixing of control and experimental samples to discover bona fide interaction partners in AP-MS experiments. We apply this intelligent mixing of proteomes (iMixPro) concept to overexpression experiments for RAF1, RNF41, and TANK and also to engineered cell lines expressing epitope-tagged endogenous PTPN14, JIP3, and IQGAP1. For all baits, we confirmed known interactions and found a number of novel interactions. The results for RNF41 and TANK were compared to a classical affinity purification experiment, which demonstrated the efficiency and specificity of the iMixPro approach.

The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions

“…Numbers indicate start and end of protein variants based on the numbering of full length (FL) human testin isoform 1 (Database ID: NP_056456.1). B) Sequence and location of testin peptides originating from endogenous testin that were identified using a mass spectrometry-based approach in the tryptic digests of complexes affinity purified using a GFP-nanobody from lysates of HeLa cells expressing ΔPET-GFP or LIM1-3-GFP as bait (PRIDE dataset identifier: PXD005058) [18,20]. …”

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

“…The workflow described above was applied to a MS dataset available on the PRIDE repository 38,39 . The original study developed a method (iMixPro), using stable isotope labeling of amino acids in cell culture (SILAC), to eliminate false positives from affinity-purification MS (AP-MS) experiments 38 . In brief, an AP-MS experiment consists of using beads-bound antibodies to fetch a protein of interest (bait) and its interactors (preys).…”

“…Here, we used SILAC to generate different isotope ratios between true preys and false positives: 3 control samples (no bait) cultured in light medium, 1 sample expressing the bait cultured in light medium, and 1 sample expressing the bait cultured in heavy medium are processed with the beads and further mass spectrometry analysis. With such design, nonspecific proteins binding to the beads will have an heavy-to-light ratio of 1:4; when true preys will have a ratio of 1:1 38 .…”

Discovering cellular protein‐protein interactions: Technological strategies and opportunities

“…Virotrap (Figure ) has a different approach for the reduction of false positives and false negatives . It eliminates the need for lysis by using virus biology.…”

“…Virotrap (Figure 7) has a different approach for the reduction of false positives and false negatives. 151 Furthermore, also similar to AVEXIS, Virotrap is able to detect membrane proteins as interactors, which is notoriously difficult for PPI technologies. However, unlike AVEXIS, the interacting prey proteins are more physiologically relevant because they are full-length, not tagged and present at the correct levels in a cellular context.…”