Affinity purification–mass spectrometry and network analysis to understand protein-protein interactions

Abstract

By determining protein-protein interactions in normal, diseased and infected cells, we can improve our understanding of cellular systems and their reaction to various perturbations. In this protocol, we discuss how to use data obtained in affinity purification–mass spectrometry (AP-MS) experiments to generate meaningful interaction networks and effective figures. We begin with an overview of common epitope tagging, expression and AP practices, followed by liquid chromatography–MS (LC-MS) data collection. We then provide a detailed procedure covering a pipeline approach to (i) pre-processing the data by filtering against contaminant lists such as the Contaminant Repository for Affinity Purification (CRAPome) and normalization using the spectral index (SIN) or normalized spectral abundance factor (NSAF); (ii) scoring via methods such as MiST, SAInt and CompPASS; and (iii) testing the resulting scores. Data formats familiar to MS practitioners are then transformed to those most useful for network-based analyses. The protocol also explores methods available in Cytoscape to visualize and analyze these types of interaction data. The scoring pipeline can take anywhere from 1 d to 1 week, depending on one’s familiarity with the tools and data peculiarities. Similarly, the network analysis and visualization protocol in Cytoscape takes 2–4 h to complete with the provided sample data, but we recommend taking days or even weeks to explore one’s data and find the right questions.

Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

“…This method combined cell lysis with a CHAPS detergent-containing buffer together with IP, SDS-PAGE-based fractionation, subsequent in-gel tryptic digestion and bottom-up mass spectrometry analysis ( Figure 1A). This approach possesses the caveat that proteins non-specifically bound to the Sepharose resin, Protein A/G, and non-targeting regions of the antibody are eluted together with the prey proteins, rendering the final interactome sample full of contaminating proteins which need systematic elimination (65,66). To avoid artefacts resulting from overexpression of epitope-tagged proteins, we used two different commercially available CLASP2 antibodies to independently IP endogenous CLASP2.…”

Analysis for Carom complex signaling and function by database mining

“…(1) Two-hybrid identify the protein-protein interaction by transcription factor activation initiated by fusion of the factor’s DNA binding domain and its transcriptional activation domain (19). (2) Affinity Capture-Mass spectrometry (MS) analysis determine the interaction when a bait protein is captured by either a specific antibody or an epitope tag and the interaction partner is analyzed via mass spectrometry (20). (3) Co-immunoprecipitation identify the interaction when a bait protein is immunoprecipitated by specific antibody and the interacting protein is also co-immunoprecipitated and identified by Western Blot (21).…”

How to Study Protein-protein Interactions

“…Data can be characterized into four classes of protein-protein interactions: biologically relevant complexes occur in the cell; physically existing interactions as artefacts of sample preparation that do not occur in the cell (e.g., interaction of proteins from different compartments); interactions involving contaminant proteins; and physically non-existing interactions detected by an error. 15 The results of TAP-MS experiments are networks. Cytoscape is a widely used tool for analyzing and visualizing these networks and a number of databases collect data from various types of protein-protein interaction experiments were launched.…”

“…Cytoscape is a widely used tool for analyzing and visualizing these networks and a number of databases collect data from various types of protein-protein interaction experiments were launched. 15 TAP-MS was successfully used to identify associated proteins to histones and new sites of post-translational modification, 16 to provide global landscape of protein complexes in the yeast Saccharomyces cerevisiae, 14 and to unravel the plant Arabidopsis protein cellular machinery complexes. 17 TAP-MS allows determination of protein partners quantitatively in vivo without prior knowledge of complex composition.…”

“…99 The basis of NMR spectroscopy is the property of many elements to have a nuclear magnetic moment. Stable isotopes of particular importance in biological macromolecules are 1 H, 13 C, or 15 N. When placed into a static magnetic field (B), the different nuclear spin states of these nuclei become quantized with energies proportional to their projection onto B. The energy difference depends on the type of nucleus, is proportional to field strength of the static magnet, and is dependent on the chemical environment of the nucleus.…”