Identifying Novel Protein‐Protein Interactions Using Co‐Immunoprecipitation and Mass Spectroscopy

Abstract

Proteomics has evolved from genomic science due to the convergence of advances in protein chemistry, separations, mass spectroscopy, and peptide and protein databases. Where identifying protein-protein interactions was once limited to yeast two-hybrid analyses or empirical data, protein-protein interactions can now be examined in both cells and native tissues by precipitation of the protein complex of interest. Coupling this field to receptor pharmacology has recently allowed for the identification of proteins that differentially and selectively interact with receptors and are integral to their biological effects. It is becoming increasingly apparent that receptors in neurons do not exist as singular independent units, but rather are part of large macromolecular complexes of interacting proteins. It is a primary quest of neuroscience to piece together these interactions and to characterize the regulatory signalplexes of all proteins. This unit presents co-immunoprecipitation-coupled mass spectroscopy as one way of identifying signalplex partners.

Cooperative interactions of LPPR/PRG family members in membrane localization and alteration of cellular morphology

“…In this study, we aimed to gain more insights into the mechanism of action of LPPR1 by identifying the LPPR1-interacting proteins using affinity purification coupled to mass spectrometry (Berggard et al, 2007;Free et al, 2009). Surprisingly, we found that another three LPPR family members, LPPR3, LPPR4 and LPPR5, were associated with LPPR1.…”

Working with Proteins in silico: A Review of Online Available Tools for Basic Identification of Proteins

“…Identifying the function of a protein in vivo, on the other hand, still requires a functional genomics approach involving detection of tissue-specific promoter activities, subcellular localization, possible post-translation modifications, and defining knock-outs or over-expressing mutants (Salzano and Crescenzi, 2005;Free et al, 2009). There are quite number of bioinformatics tools that can be used for in silico analysis of a protein to design a functional genomics approach (Table 1), thereby, in this manuscript we aimed to summarize online available tools for characterization of the protein of interest through in silico calculation of molecular weight and isoelectric point, detection of protein motifs and domains, estimation of subcellular localization and possible post-translational modifications.…”

Biochemical & Biophysical Studies of an Hfq Homolog from a Deep-branching Bacterium

“…The bacterial Hfq interacts with both A-rich mRNAs and uridine-rich (U-rich) sRNAs on opposite faces of the discshaped Hfq hexamer, referred to as the "distal" and "proximal" face, respectively [66]. The specific population of RNAs bound to Hfq can vary significantly between different homologs [12,28,67]. In terms of physiological effects, the Hfq protein decreases the rate of RNA turnover of bound RNAs and facilitates interactions between sRNAs and their target mRNAs.…”

“…A second model suggests that RNA binds to two separate Hfq hexamers and interaction between the two hexamers facilitates annealing [67]. This model is partially supported by the crystal structure of AU 6 A with Hfq, which revealed the AU 6 A bound to the proximal face of one Hfq with the 5' adenosine docked to the distal face of another Hfq ring [82].…”

“…Proteins and RNAs physically associated with Hfq can be, in principle, isolated by co-immunoprecipitation using anti-Hfq pAbs [51,52]. The protein interacting partners co-immunoprecipitate with Hfq can then be identified by well-established proteomics experiments [65,66,67]. Immunoprecipitated RNA components can be further purified from the protein using phenol-chloroform extraction based technologies and identified using next-generation sequencing [3,53,54,55].…”