Isolation of signal transduction complexes using biotin and crosslinking methodologies

Abstract

We have developed a strategy to preferentially label the N-terminal a-amino groups of intact proteins allowing the internal e-amino groups to remain free to react with chemical crosslinking reagents. The convergence of these methodologies allows biotinylated ligands to bind to their receptors within the cell membrane followed by removal of the crosslinked complex from cell lysate. This technique allows for the isolation of protein complexes in an MS-compatible system, thus providing a tool for furthering our understanding of signal transduction. Keywords:Biotin / Crosslinking / Signaling / TNF-alpha Proteomics 2007Proteomics , 7, 2371Proteomics -2374 2371The emerging field of functional proteomics has facilitated the study of protein networks, interactions within the cell due to stimulation or perturbation of the cellular environment [1]. The ability to isolate signaling complexes can not only provide information regarding the components of the complex, but also aid a more complete understanding of how these components are altered in disease states. Numerous strategies have been reported to isolate multiprotein complexes followed by identification of the components of the complex by MS methods. The majority of isolation strategies utilize affinity-based techniques that require the overexpression of a fusion protein within the cell that is later immunoprecipitated by an antibody against its epitope thus allowing for the isolation of the target protein and interacting partners [2]. Methods such as tandem affinity purification (TAP) tags [3] utilize this principle and have been successful in the identification of protein assemblies. While the isolation of macromolecular complexes provides insight into regulated cellular processes, there exists a vast interest in protein complex formation due to extracellular signals [4]. While endogenous complexes are critical for cellular signaling, quite often the need for signaling is provided by an extracellular molecule that triggers a cellular response following binding to a specific receptor within the plasma membrane.Biotin has been used extensively as an affinity fusion partner due to its remarkably strong interaction with streptavidin (K d ,10 215 7M) [5]. Appropriate addition of biotin to a recombinant protein can eliminate concerns commonly encountered with the production of fusion proteins; for example protein folding, interruption of protein activity or function, yield, and solubility. Preferential biotinylation of proteins at the N-terminus maintains the integrity of the binding site, allowing the baited protein to interact with the appropriate receptor and initiate a signal throughout the cell [6]. With this approach, lysine residues containing primary amines remain available for reaction with crosslinker reagents, chemicals commonly used to study protein-protein interactions [7]. We report here a new strategy to isolate signaling complexes through combination of biotin addition and crosslinking methodologies. In contrast to other approaches, the external signal,...

Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress

“…Preferential NH 2 -terminal biotin labeling of a bait protein, avoiding multiple labels potentially blocking binding, has been shown to work efficiently in previous studies (26,27,41); however, a free NH 2 terminus is required. VEGF-A does not contain a free NH 2 terminus, hence is not amenable for NH 2 -terminal biotin labeling; thus a VEGF-A-coupled metallic epoxy resin was used in this study with cell-permeable cross-linkers to compensate (26,27,35,44). However, even with the ability to cross-link protein signaling complexes there is still great difficulty in efficiently and effectively isolating these cross-linked complexes from the tissue or cell sample.…”

“…The emergence of microarray technologies leading to high-throughput discovery of novel gene expression changes has been beneficial in building signaling pathways (42); however, the measurement of RNA expression is still a step removed from protein pathways critical for signaling. To overcome this caveat, proteomics has advanced over recent years to allow development of highthroughput discovery studies through utilization of advanced technologies in exploring cascades of signaling events by analyzing cellular phosphoproteome, protein-protein interactions, DNA-protein interactions, and quantitative protein abundance under varying conditions or disease models (11,26,27,35,41,44,46,48). Methods such as immunoisolation and protein biotin labeling to purify protein signaling complexes, followed by MS analysis have been commonly used to explore signaling pathways (26,27,41); however, these methods are not ideal in all conditions.…”

“…1). The use of various cross-linkers coupled with tandem MS analysis is becoming increasingly common in the study of protein-protein interactions and protein pathway analysis (26,27,35,44). We hypothesized that hypoxic conditions, simulating cardiovascular stress, would enhance VEGF-A signaling in BM-EPCs compared with the normoxic state.…”

Overcoming Key Technological Challenges in Using Mass Spectrometry for Mapping Cell Surfaces in Tissues

“…Although the method appears intuitive, there may be problems relating to the lysis of the cells by the proteases, thus contaminating the membrane "peptide" solution with intracellular proteins. An alternative approach is the cross-linking of PM protein complexes where specific reagents are used to maintain protein complexes in their close to native state (18,19). The cells are subsequently lysed, and the non-complexed proteins can be removed by various methods, including size exclusion chromatography.…”

Application of Two-Dimensional Gel Electrophoresis to Microbial Systems

“…Proteomics is regarded as a powerful approach as far as biochemical research is concerned, because it directly studies the key functional components of biochemical systems, namely proteins (Freed et al, 2007). Approximately 70% of a microbial cell is composed of proteins (Frantz & Maccallum, 1980) and can aid in discriminating and distinguishing between different species and different bacterial strains (Fagerquist et al, 2005).…”