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Proteome of the Escherichia Coli Envelope and Technological Challenges in Membrane Proteome Analysis ⁎ Joel H

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Proteome of the Escherichia Coli Envelope and Technological Challenges in Membrane Proteome Analysis ⁎ Joel H View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 1698–1713 www.elsevier.com/locate/bbamem Review Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis ⁎ Joel H. Weiner a,b, , Liang Li c a Membrane Protein Research Group and The Institute for Biomolecular Design, University of Alberta, Canada b Department of Biochemistry and Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta, Canada T6G 2E7 c Department of Chemistry and The Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta, Canada T6G 2E1 Received 18 April 2007; received in revised form 19 July 2007; accepted 23 July 2007 Available online 11 August 2007 Abstract The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, β-sheet motifs and stretches of α- helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane- associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem. © 2007 Elsevier B.V. All rights reserved. Keywords: Cytoplasmic membrane; Periplasm; Outer membrane; Mass spectrometry; Proteomics; Polyacrylamide gel electrophoresis Contents 1. Introduction ............................................................. 1699 2. E. coli envelope composition .................................................... 1699 3. The outer membrane proteome ................................................... 1700 3.1. Predicted outer membrane proteome ............................................. 1700 3.2. Experimental outer membrane proteome ........................................... 1701 4. The periplasmic proteome ...................................................... 1702 4.1. Predicted periplasmic proteome ............................................... 1702 4.2. Experimental periplasmic proteome ............................................. 1703 Abbreviations: 2MEGA, dimethylation after lysine guanidination; ABC, ATP binding cassette; CNBr, cyanogen bromide; ESI, electrospray ionization; ICAT, isotope-coded affinity tag; IEF, isoelectric focusing; IMP, inner membrane protein; LC, liquid chromatography; MAAH, microwave-assisted acid hydrolysis; MALDI- TOF, matrix assisted laser desorption/ionization-time of flight; MS, mass spectrometry; OMP, outer membrane protein; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PMF, peptide mass fingerprinting; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid; TMM, transmembrane ⁎ Corresponding author. Department of Biochemistry and Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Tel.: +1 780 492 2761; fax: +1 780 492 0886. E-mail addresses: [email protected] (J.H. Weiner), [email protected] (L. Li). 0005-2736/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2007.07.020 J.H. Weiner, L. Li / Biochimica et Biophysica Acta 1778 (2008) 1698–1713 1699 5. The cytoplasmic membrane proteome ................................................1704 5.1. Predicted cytoplasmic membrane proteome ..........................................1704 5.2. Respiratory complexes.....................................................1705 5.3. Experimental cytoplasmic membrane proteome ........................................1705 6. Comparison with microarray .....................................................1706 7. Methodology for membrane proteome analysis............................................1706 7.1. Gel-based proteome analysis platform.............................................1706 7.2. Solution-based proteome analysis platform ..........................................1708 8. Future perspectives ..........................................................1710 Acknowledgements ............................................................1710 References .................................................................1710 1. Introduction cellular proteome is associated with the envelope and at least one half of these proteins are of unknown or putative function. It is The availability of complete genomic sequences for hundreds quite possible that the functionally uncharacterized proteins of microorganisms, as well as transcriptome measurements of catalyze unique metabolic transitions and these may prove mRNA levels under a variety of physiologic conditions, has useful for the biotechnology industry. Thus, a full understanding spurred investigators to determine the complete protein compo- of E. coli requires the development of novel technologies sition, or proteome, of entire bacterial cells and how the pro- including proteomic analysis to determine the function of these teome changes with physiologic shifts. Membrane proteins proteins. Proteomic studies under varying growth, regulatory which play essential roles in energetics, metabolism, signal and stress conditions will help with the functional assignment of transduction and transport compose as much as 40% of the unknown proteins. In addition, because of the hydrophobicity of entire proteome of a bacterial cell. The hydrophobic nature of membrane proteins there are significant technical difficulties in membrane proteins has hindered their investigation at the studying the membrane proteome and E. coli provides a standard functional and structural level. Similarly, characterizing the for evaluating and validating new technologies. E. coli offers an membrane proteome has lagged studies of the soluble prote- excellent model system to develop new extraction, solubiliza- ome. However, recent technical advances in separation metho- tion, proteolytic, electrophoretic and mass spectrometry techni- dologies by 2-dimensional electrophoretic techniques and 2- ques which will be applied to the more complex proteomes of dimensional HPLC methodologies, coupled with improved higher organisms. proteolytic methodologies, and advanced mass spectrometry instrumentation is opening the membrane proteome to experi- 2. E. coli envelope composition mental evaluation. This review will summarize current data on the predicted and The envelope of E. coli is a complex structure composed of observed proteome of the Escherichia coli K-12 envelope along the outer membrane, periplasm/peptidoglycan layer and the with advances in methodology that allow more detailed ex- cytoplasmic membrane. The envelope is usually prepared by ploration of this proteome. lysozyme-EDTA lysis [3], French press lysis [4] or Avestin The complete proteome of E. coli is being examined for EmulsiFlex [5] lysis of bacterial cultures. These processes form many reasons. The International E. coli Alliance [1] brings vesicles that can be isolated by differential centrifugation. Inner together biochemists, system biologists and computer scientists and outer membranes can be isolated by density centrifugation with the aim of developing a detailed understanding of the entire comprising an 8000×g spin to sediment unbroken cells and transcriptome, proteome, interactome, metabolome and phy- debris followed by a high speed spin (150,000×g) to sediment siome of E. coli in response to physiologic, genetic or environ- inner and outer membrane vesicles [6]. Inner from outer mem- mental variation with the aim of creating an in silico E. coli brane vesicles can then be separated by density centrifugation where changes can be predicted and experimentally verified. using a sucrose gradient, with the outer membranes having a Whereas the transcriptome determined by microarray techni- higher density. In most cases vesicle integrity is such that ques gives a whole genome profile at the mRNA level, pro- cytosolic proteins trapped in the vesicles can be removed by teomics gives information on expression levels. Proteomics buffer washing. Loosely-associated extrinsic proteins are often provides information on post-translational modifications which removed by washing with chaotropic agents such as Na car- is not possible by microarray and proteomics can be used to bonate [7]. Fractions containing
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