The Escherichia coli Proteome: Past, Present, and Future Prospects

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects.     

Characterizing phosphoproteins and phosphoproteomes using mass spectrometry.

The reversible phosphorylation of proteins plays a major role in many vital cellular processes by modulating protein function and transmitting signals within cellular pathways and networks. Because phosphorylation is dynamic and the sites of modification cannot be predicted by an organism's genome, proteomic measurements are required to identify sites of and changes in the phosphorylation state of proteins. The low stoichiometry of phosphorylation sites that accompany the multifarious nature of protein phosphorylation in biological systems continues to challenge the dynamic range of present mass spectrometry (MS) technologies and proteomic measurements, despite the preponderance of research and analytical methods devoted to this area. This review addresses some of the strategies and limitations involving the use of MS to map and quantify changes in protein phosphorylation sites for samples that range from a single protein to an entire proteome, and presents several compelling reasons as to why comprehensive phosphorylation site analysis has proven to be so elusive without a hypothesis-driven experimental approach to elicit more meaningful and confident results.     

Gel-free mass spectrometry-based high throughput proteomics: tools for studying biological response of proteins and proteomes

Revolutionary advances in biological mass spectrometry (MS) have provided a basic tool to make possible comprehensive proteomic analysis. Traditionally, two-dimensional gel electrophoresis has been used as a separation method coupled with MS to facilitate analysis of complex protein mixtures. Despite the utility of this method, the many challenges of comprehensive proteomic analysis has motivated the development of gel-free MS-based strategies to obtain information not accessible using two-dimensional gel separations. These advanced strategies have enabled researchers to dig deeper into complex proteomes, gaining insights into the composition, quantitative response, covalent modifications and macromolecular interactions of proteins that collectively drive cellular function. This review describes the current state of gel-free, high throughput proteomic strategies using MS, including (i) the separation approaches commonly used for complex mixture analysis; (ii) strategies for large-scale quantitative analysis; (iii) analysis of post-translational modifications; and (iv) recent advances and future directions. The use of these strategies to make new discoveries at the proteome level into the effects of disease or other cellular perturbations is discussed in a variety of contexts, providing information on the potential of these tools in electromagnetic field research.     

Web-accessible proteome databases for microbial research

The analysis of proteomes of biological organisms represents a major challenge of the post-genome era. Classical proteomics combines two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) for the identification of proteins. Novel technologies such as isotope coded affinity tag (ICAT)-liquid chromatography/mass spectrometry (LC/MS) open new insights into protein alterations. The vast amount and diverse types of proteomic data require adequate web-accessible computational and database technologies for storage, integration, dissemination, analysis and visualization. A proteome database system (http://www.mpiib-berlin.mpg.de/2D-PAGE) for microbial research has been constructed which integrates 2-DE/MS, ICAT-LC/MS and functional classification data of proteins with genomic, metabolic and other biological knowledge sources. The two-dimensional polyacrylamide gel electrophoresis database delivers experimental data on microbial proteins including mass spectra for the validation of protein identification. The ICAT-LC/MS database comprises experimental data for protein alterations of mycobacterial strains BCG vs. H37Rv. By formulating complex queries within a functional protein classification database "FUNC_CLASS" for Mycobacterium tuberculosis and Helicobacter pylori the researcher can gather precise information on genes, proteins, protein classes and metabolic pathways. The use of the R language in the database architecture allows high-level data analysis and visualization to be performed "on-the-fly". The database system is centrally administrated, and investigators without specific bioinformatic competence in database construction can submit their data. The database system also serves as a template for a prototype of a European Proteome Database of Pathogenic Bacteria. Currently, the database system includes proteome information for six strains of microorganisms.     

Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.     

Tissue proteomics using chemical immobilization and mass spectrometry

Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.     

Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data.Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared.This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.     

Utility of mass spectrometry for proteome analysis: part II. Ion-activation methods,statistics, bioinformatics and annotation

This is the second article in a series, intended as a tutorial to provide the interested reader with an overview of the concepts not covered in part I, such as: the principles of ion-activation methods, the ability of mass-spectrometric methods to interface with various proteomic strategies, analysis techniques, bioinformatics and data interpretation and annotation. Although these are different topics, it is important that a reader has a basic and collective understanding of all of them for an overall appreciation of how to carry out and analyze a proteomic experiment. Different ion-activation methods for MS/MS, such as collision-induced dissociation (including postsource decay) and surface-induced dissociation, electron capture and electron-transfer dissociation, infrared multiphoton and blackbody infrared radiative dissociation have been discussed since they are used in proteomic research. The high dimensionality of data generated from proteomic studies requires an understanding of the underlying analytical procedures used to obtain these data, as well as the development of improved bioinformatics tools and data-mining approaches for efficient and accurate statistical analyses of biological samples from healthy and diseased individuals, in addition to determining the utility of the interpreted data. Currently available strategies for the analysis of the proteome by mass spectrometry, such as those employed for the analysis of substantially purified proteins and complex peptide mixtures, as well as hypothesis-driven strategies, have been elaborated upon. Processing steps prior to the analysis of mass spectrometry data, statistics and the several informatics steps currently used for the analysis of shotgun proteomic experiments, as well as proteomics ontology, are also discussed.     

Cellular metabolomics of Escherchia coli

Escherichia coli is among the simplest and best-understood free-living organisms. It has served as a valuable model for numerous biological processes, including cellular metabolism. Just as E. coli stood at the front of the genomic revolution, it is playing a leading role in the development of cellular metabolomics: the study of the complete metabolic contents of cells, including their dynamic concentration changes and fluxes. This review briefly describes the essentials of cellular metabolomics and its fundamental differentiation from biomarker metabolomics and lipidomics. Key technologies for metabolite quantitation from E. coli are described, with a focus on those involving mass spectrometry. In particular emphasis is given to the cell handling and sample preparation steps required for collecting data of high biological reliability, such as fast metabolome quenching. Future challenges, both in terms of data collection and application of the data to obtain a comprehensive understanding of metabolic dynamics, are discussed.     

Arabidopsis thaliana proteomics: from proteome to genome

Proteomics has become an important approach for investigating cellular processes and network functions. Significant improvements have been made during the last few years in technologies for high-throughput proteomics, both at the level of data analysis software and mass spectrometry hardware. As proteomics technologies advance and become more widely accessible, efforts of cataloguing and quantifying full proteomes are underway to complement other genomics approaches, such as RNA and metabolite profiling. Of particular interest is the application of proteome data to improve genome annotation and to include information on post-translational protein modifications with the annotation of the corresponding gene. This type of analysis requires a paradigm shift because amino acid sequences must be assigned to peptides without relying on existing protein databases. In this review, advances and current limitations of full proteome analysis are briefly highlighted using the model plant Arabidopsis thaliana as an example. Strategies to identify peptides are also discussed on the basis of MS/MS data in a protein database-independent approach.     

Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis

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, beta-sheet motifs and stretches of alpha-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.     

Biomarker discovery: proteome fractionation and separation in biological samples.

Proteomics, analogous with genomics, is the analysis of the protein complement present in a cell, organ, or organism at any given time. While the genome provides information about the theoretical status of the cellular proteins, the proteome describes the actual content, which ultimately determines the phenotype. The broad application of proteomic technologies in basic science and clinical medicine has the potential to accelerate our understanding of the molecular mechanisms underlying disease and may facilitate the discovery of new drug targets and diagnostic disease markers. Proteomics is a rapidly developing and changing scientific discipline, and the last 5 yr have seen major advances in the underlying techniques as well as expansion into new applications. Core technologies for the separation of proteins and/or peptides are one- and two-dimensional gel electrophoresis and one- and two-dimensional liquid chromatography, and these are coupled almost exclusively with mass spectrometry. Proteomic studies have shown that the most effective analysis of even simple biological samples requires subfractionation and/or enrichment before protein identification by mass spectrometry. Selection of the appropriate technology or combination of technologies to match the biological questions is essential for maximum coverage of the selected subproteome and to ensure both the full interpretation and the downstream utility of the data. In this review, we describe the current technologies for proteome fractionation and separation of biological samples, based on our lab workflow for biomarker discovery and validation.     

Role of chromatographic techniques in proteomic analysis

Proteomics, the characterization of the proteome, is conceptually simple but technically challenging. Development of such technologies as mass spectrometry, multidimensional protein separation, and DNA sequencing has allowed the new field of proteomics to flourish. Proteomic analysis relies on a set of techniques chosen on the basis of the biological question. In any proteomic analysis, the first and most important task is the separation of a complex protein mixture, i.e. the proteome. Chromatography, one of the most powerful methods of separation, employs one or more inherent characteristics of a protein-its mass, isoelectric point, hydrophobicity or biospecificity. This review emphasizes high-performance liquid chromatography as an integrated part of technologies used to study the proteome, discusses the capabilities and limitations of current instruments, and highlights the potential of multidimensional liquid chromatography in proteomic analysis.     

Escherichia coli--a model system that benefits from and contributes to the evolution of proteomics

The large body of knowledge about Escherichia coli makes it a useful model organism for the expression of heterologous proteins. Proteomic studies have helped to elucidate the complex cellular responses of E. coli and facilitated its use in a variety of biotechnology applications. Knowledge of basic cellular processes provides the means for better control of heterologous protein expression. Beyond such important applications, E. coli is an ideal organism for testing new analytical technologies because of the extensive knowledge base available about the organism. For example, improved technology for characterization of unknown proteins using mass spectrometry has made two-dimensional electrophoresis (2DE) studies more useful and more rewarding, and much of the initial testing of novel protocols is based on well-studied samples derived from E. coli. These techniques have facilitated the construction of more accurate 2DE maps. In this review, we present work that led to the 2DE databases, including a new map based on tandem time-of-flight (TOF) mass spectrometry (MS); describe cellular responses relevant to biotechnology applications; and discuss some emerging proteomic techniques.     

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.     

Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry.

Proteomics has been proposed as one of the key technologies in the postgenomic era. So far, however, the comprehensive analysis of cellular proteomes has been a challenge because of the dynamic nature and complexity of the multitude of proteins in cells and tissues. Various approaches have been established for the analyses of proteins in a cell at a given state, and mass spectrometry (MS) has proven to be an efficient and versatile tool. MS-based proteomics approaches have significantly improved beyond the initial identification of proteins to comprehensive characterization and quantification of proteomes and their posttranslational modifications (PTMs). Despite these advances, there is still ongoing development of new technologies to profile and analyze cellular proteomes more completely and efficiently. In this review, we focus on MS-based techniques, describe basic approaches for MS-based profiling of cellular proteomes and analysis methods to identify proteins in complex mixtures, and discuss the different approaches for quantitative proteome analysis. Finally, we briefly discuss novel developments for the analysis of PTMs. Altered levels of PTM, sometimes in the absence of protein expression changes, are often linked to cellular responses and disease states, and the comprehensive analysis of cellular proteome would not be complete without the identification and quantification of the extent of PTMs of proteins.     

Proteomic reactors and their applications in biology

Proteomic analysis requires the combination of an extensive suite of technologies including protein processing and separation, micro-flow HPLC, MS and bioinformatics. Although proteomic technologies are still in flux, approaches that bypass gel electrophoresis (gel-free approaches) are dominating the field of proteomics. Along with the development of gel-free proteomics, came the development of devices for the processing of proteomic samples termed proteomic reactors. These microfluidic devices provide rapid, robust and efficient pre-MS sample procession by performing protein sample preparation/concentration, digestion and peptide fractionation. The proteomic reactor has advanced in two major directions: immobilized enzyme reactor and ion exchange-based proteomic reactor. This review summarizes the technical developments and biological applications of the proteomic reactor over the last decade.