A “Tagless” Strategy for Identification of Stable Protein Complexes Genome-wide by Multidimensional Orthogonal Chromatographic Separation and iTRAQ Reagent Tracking

Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Journal of Proteome Research (Impact Factor: 4.25). 06/2008; 7(5):1836-49. DOI: 10.1021/pr700624e
Source: PubMed


Tandem affinity purification is the principal method for purifying and identifying stable protein complexes system-wide in whole cells. Although highly effective, this approach is laborious and impractical in organisms where genetic manipulation is not possible. Here, we propose a novel "tagless" strategy that combines multidimensional separation of endogenous complexes with mass spectrometric monitoring of their composition. In this procedure, putative protein complexes are identified based on the comigration of collections of polypeptides through multiple orthogonal separation steps. We present proof-of-principle evidence for the feasibility of key aspects of this strategy. A majority of Escherichia coli proteins are shown to remain in stable complexes during fractionation of a crude extract through three chromatographic steps. We also demonstrate that iTRAQ reagent-based tracking can quantify relative migration of polypeptides through chromatographic separation media. LC MALDI MS and MS/MS analysis of the iTRAQ-labeled peptides gave reliable relative quantification of 37 components of 13 known E. coli complexes: 95% of known complex components closely co-eluted and 57% were automatically grouped by a prototype computational clustering method. With further technological improvements in each step, we believe this strategy will dramatically improve the efficiency of the purification and identification of protein complexes in cells.

29 Reads
  • Source
    • "Importantly, TAP is not possible in most crop plants because it is not feasible to generate the enormous number of high-quality transgenic lines that would be needed. An alternative approach to protein complex analysis is to capitalize on the parallel protein detection and quantification capability of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze endogenous protein complexes (Dong et al., 2008; Liu et al., 2008). In plants, the potential benefits of label-free MS protein quantification have been discussed (Thelen and Peck, 2007), and these methods have been used to analyze the size and putative composition of endogenous protein complexes in the chloroplast stroma that were separated by native gel electrophoresis (Peltier et al., 2006) and size exclusion chromatography (SEC) (Olinares et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast-two-hybrid approaches. These methods are time consuming, and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a new method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves, and combines chromatographic separation of extracts from sub-cellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by western blotting, and comparisons with published size exclusion chromatography data and the masses of known complexes. This method is simple to implement with appropriate instrumentation and is applicable to any biological system. It has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.
    The Plant Cell 09/2014; 26(10). DOI:10.1105/tpc.114.127563 · 9.34 Impact Factor
  • Source
    • "However, many of these complexes have low copy numbers inside cells. Different techniques have been developed to enrich them for experimental investigations (Dong et al., 2008). We are interested in engineering a selection surface that allows affinity-based retention of macromolecular complexes in high density. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and chemical linkage to introduce a high density of bioactive ligands onto nanometer-thick carbon films and enable selective enrichment of individual macromolecular complexes at subnanogram levels. The introduced ligands are physically separated. Ni-NTA, Protein G and DNA/RNA oligonucleotides were covalently linked to the carbon surface. They embody negligible mass and their stability makes the functionalized films able to survive long-term storage and tolerate variations in pH, temperature, salts, detergents, and solvents. We demonstrated the application of the new method to the electron microscopic imaging of the substrate-bound C3PO, an RNA-processing enzyme important for the RNA interference pathway. On the ssRNA-linked carbon surface, the formation of C3PO oligomers at subnanomolar concentrations likely mimics their assembly onto ssRNA substrates presented by their native partners. Interestingly, the 3D reconstructions by negative stain EM reveal a side port in the C3PO/ssRNA complex, and the 15 Å cryoEM map showed extra density right above the side port, which probably represents the ssRNA. These results suggest a new way for ssRNAs to interact with the active sites of the complex. Together our data demonstrate that the surface-engineered carbon films are suitable for selectively enriching low-abundance biological complexes at nanomolar level and for developing novel applications on a large number of surface-presented molecules.
    Journal of Structural Biology 01/2014; 185(3). DOI:10.1016/j.jsb.2014.01.006 · 3.23 Impact Factor
  • Source
    • "However, many of these complexes have low copy numbers inside cells. Different techniques have been developed to enrich them for experimental investigations (Dong et al., 2008). We are interested in engineering a selection surface that allows affinity-based retention of macromolecular complexes in high density. "
    Biophysical Journal 01/2012; 102(3):394-. DOI:10.1016/j.bpj.2011.11.2151 · 3.97 Impact Factor
Show more