Approaches to Top-Down Proteomics: In Honour of Prof. Patrick H. O'Farrell

A special issue of Proteomes (ISSN 2227-7382).

Deadline for manuscript submissions: closed (30 September 2016) | Viewed by 91952

Special Issue Editors

1. Department of Biological Sciences, Faculty of Mathematics and Science, Brock University, St. Catharines, ON L2S 3A1, Canada
2. Ronin Institute, Montclair, NJ 07043, USA
3. Institute for Globally Distributed Open Research and Education (IGDORE), Catharines, ON L2S 3A1, Canada
Interests: analytical proteomics; high-resolution discovery proteomics; top-down proteomics; two-dimensional gel electrophoresis; 2DE/MS/MS; proteoforms/protein species; molecular mechanisms; biomarkers; membrane proteomes; synaptic function; lipidomics; metabolomics; translational proteomics; systems biology; exocytosis; preterm labor; spinal cord injury; neurodegenerative disorders
Special Issues, Collections and Topics in MDPI journals
NIH, Building 10, Room 9D 52, Bethesda, MD 20892, USA
Interests: 2DE; bottom-up; discovery proteomics; LC/MS/MS; protein species; proteoforms, top-down; de novo sequencing; novel data analysis approaches

Special Issue Information

Dear Colleagues,

Presaging the current discipline of Proteomics, Prof Patrick H. O’Farrell* recognized the critical need for detailed protein analyses to dissect and thereby understand molecular mechanisms. His development of two-dimensional gel electrophoresis (2DE) has proven a cornerstone of modern protein science and, together with developments in mass spectrometry and genomics, laid the groundwork for current Systems Biology approaches to understanding the molecular basis of physiological states in health and disease. Notably, as refined over the last 40 years, 2DE provides the only currently available, optimized, routine, Top-down analytical approach that resolves proteoforms (i.e., protein species including isoforms and post-translationally modified variants)—those protein entities that define biological functions. The future clearly holds even more promise in terms of the further development of both Discovery and Targeted Top-down Proteomics to understanding biological pathways and mechanisms.

Thus, we are soliciting for interest and suggestions for manuscripts for this Special Issue (SI). In addition to an already commissioned historical perspective reviewing gel electrophoresis, immunoblotting, pull-downs, and so forth, we plan for this SI to include papers on current top-down Discovery approaches (e.g., modern gel-based/gel-free techniques and MS-intensive analytical approaches as the future), as well as Targeted Proteomics approaches (e.g. antibody arrays/protein atlas approaches, QconCAT, proximity/hybridization-based methods, and others). The overarching criteria for final acceptance of any manuscript will be critical reasoning and rigor; presenting and examining the genuine pros and cons of different analytical approaches in order to yield a SI of broad interest as a teaching tool and analytical resource for students through to experienced researchers. We expect this approach to help revise much of the dogma that has arisen in the literature over the last 20+ years with regard to Top-down Proteomics and to illuminate promising directions in further dissecting and understanding molecular mechanisms and thus cellular processes. We will be seeking a balance between review articles and original scientific reports, with the latter being favored (or a combined approach reviewing the field and presenting original data concerning the analytical approach in focus).

Prof. Dr. Jens R. Coorssen
Dr. Alfred L. Yergey
Guest Editors

*Prof. O’Farrell is a leading molecular biologist who made crucial contributions to the development of 2-dimensional protein electrophoresis (2DE). He graduated from McGill University (Montreal) and did his graduate work in the University of Colorado, where he developed the technique of 2DE. In 1979 he joined the Department of Biochemistry at UCSF and became one of the pioneers of molecular analysis of embryonic pattern formation. He identified the gene encoding the homeodomain protein Engrailed and his analysis of its function in segmentation defined processes used throughout the regulatory cascade that patterns the embryo. He turned to study the control of the cell cycle during development using Drosophila genetics, molecular biology and imaging. Recent work has demonstrated the importance of regulation of the S phase duration in these events. A newer project area explores the biology and genetics of the mitochondrial genome.

Manuscript Submission Information

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Top-down Proteomics
  • Proteoforms
  • protein species
  • two-dimensional gel electrophoresis
  • Discovery Proteomics
  • Targeted Proteomics
  • CE-MS of Intact Proteins

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Published Papers (12 papers)

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Editorial

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164 KiB  
Editorial
Editorial for Special Issue: Approaches to Top-Down Proteomics: In Honour of Prof. Patrick H. O’Farrell
by Jens Coorssen and Alfred Yergey
Proteomes 2017, 5(3), 18; https://doi.org/10.3390/proteomes5030018 - 24 Jul 2017
Cited by 3 | Viewed by 3142
Abstract
Presaging the current discipline of Proteomics, Prof Patrick H. O’Farrell recognized the critical need for detailed protein analyses to dissect and thereby understand molecular mechanisms. [...]
Full article

Research

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4669 KiB  
Article
A Routine ‘Top-Down’ Approach to Analysis of the Human Serum Proteome
by Arlene M. D’Silva, Jon A. Hyett and Jens R. Coorssen
Proteomes 2017, 5(2), 13; https://doi.org/10.3390/proteomes5020013 - 06 Jun 2017
Cited by 14 | Viewed by 5872
Abstract
Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address [...] Read more.
Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address this, these can lead to the concomitant removal of non-targeted protein species, and thus raise issues of specificity, reproducibility, and the capacity for meaningful quantitative analyses. Altering the native stoichiometry of the proteome components may thus yield a more complex series of issues than dealing directly with the inherent complexity of the sample. Hence, here we targeted method refinements so as to ensure optimum resolution of serum proteomes via a top down two-dimensional gel electrophoresis (2DE) approach that enables the routine assessment of proteoforms and is fully compatible with subsequent mass spectrometric analyses. Testing included various fractionation and non-fractionation approaches. The data show that resolving 500 µg protein on 17 cm 3–10 non-linear immobilised pH gradient strips in the first dimension followed by second dimension resolution on 7–20% gradient gels with a combination of lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS) detergents markedly improves the resolution and detection of proteoforms in serum. In addition, well established third dimension electrophoretic separations in combination with deep imaging further contributed to the best available resolution, detection, and thus quantitative top-down analysis of serum proteomes. Full article
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278 KiB  
Article
De Novo Sequencing of Top-Down Tandem Mass Spectra: A Next Step towards Retrieving a Complete Protein Sequence
by Kira Vyatkina
Proteomes 2017, 5(1), 6; https://doi.org/10.3390/proteomes5010006 - 08 Feb 2017
Cited by 14 | Viewed by 5785
Abstract
De novo sequencing of tandem (MS/MS) mass spectra represents the only way to determine the sequence of proteins from organisms with unknown genomes, or the ones not directly inscribed in a genome—such as antibodies, or novel splice variants. Top-down mass spectrometry provides new [...] Read more.
De novo sequencing of tandem (MS/MS) mass spectra represents the only way to determine the sequence of proteins from organisms with unknown genomes, or the ones not directly inscribed in a genome—such as antibodies, or novel splice variants. Top-down mass spectrometry provides new opportunities for analyzing such proteins; however, retrieving a complete protein sequence from top-down MS/MS spectra still remains a distant goal. In this paper, we review the state-of-the-art on this subject, and enhance our previously developed Twister algorithm for de novo sequencing of peptides from top-down MS/MS spectra to derive longer sequence fragments of a target protein. Full article
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8767 KiB  
Article
Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome
by Kathrin Kusch, Marina Uecker, Thomas Liepold, Wiebke Möbius, Christian Hoffmann, Heinz Neumann, Hauke B. Werner and Olaf Jahn
Proteomes 2017, 5(1), 3; https://doi.org/10.3390/proteomes5010003 - 12 Jan 2017
Cited by 14 | Viewed by 6704
Abstract
Post-translational modifications (PTMs) play a key role in regulating protein function, yet their identification is technically demanding. Here, we present a straightforward workflow to systematically identify post-translationally modified proteins based on two-dimensional gel electrophoresis. Upon colloidal Coomassie staining the proteins are partially transferred, [...] Read more.
Post-translational modifications (PTMs) play a key role in regulating protein function, yet their identification is technically demanding. Here, we present a straightforward workflow to systematically identify post-translationally modified proteins based on two-dimensional gel electrophoresis. Upon colloidal Coomassie staining the proteins are partially transferred, and the investigated PTMs are immunodetected. This strategy allows tracking back the immunopositive antigens to the corresponding spots on the original gel, from which they are excised and mass spectrometrically identified. Candidate proteins are validated on the same membrane by immunodetection using a second fluorescence channel. We exemplify the power of partial immunoblotting with the identification of lysine-acetylated proteins in myelin, the oligodendroglial membrane that insulates neuronal axons. The excellent consistency of the detected fluorescence signals at all levels allows the differential comparison of PTMs across multiple conditions. Beyond PTM screening, our multi-level workflow can be readily adapted to clinical applications such as identifying auto-immune antigens or host-pathogen interactions. Full article
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2066 KiB  
Article
Targeted Enlargement of Aptamer Functionalized Gold Nanoparticles for Quantitative Protein Analysis
by Feng Li, Jingjing Li, Yanan Tang, Chuan Wang, Xing-Fang Li and X. Chris Le
Proteomes 2017, 5(1), 1; https://doi.org/10.3390/proteomes5010001 - 22 Dec 2016
Cited by 15 | Viewed by 5614
Abstract
The ability to selectively amplify the detection signals for targets over interferences is crucial when analyzing proteins in a complicated sample matrix. Here, we describe a targeted enlargement strategy that can amplify the light-scattering signal from aptamer-functionalized gold nanoparticles (Apt-AuNP) with high specificity [...] Read more.
The ability to selectively amplify the detection signals for targets over interferences is crucial when analyzing proteins in a complicated sample matrix. Here, we describe a targeted enlargement strategy that can amplify the light-scattering signal from aptamer-functionalized gold nanoparticles (Apt-AuNP) with high specificity for quantitative protein analysis. This strategy is achieved by labeling target proteins with competitively protected Apt-AuNP probes and enlarging the probes with gold enhancement. This competitive protection strategy could effectively eliminate nonspecific protein adsorptions from a sample matrix, leading to a highly specific labeling of the target protein. As a result, the subsequent amplification of the light-scattering signal by gold enhancement only occurs in the presence of the target protein. This strategy was successfully demonstrated by analyzing human α-thrombin in human serum samples in a Western blot format. Full article
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Review

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771 KiB  
Review
A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis
by Matthew P. Padula, Iain J. Berry, Matthew B. O′Rourke, Benjamin B.A. Raymond, Jerran Santos and Steven P. Djordjevic
Proteomes 2017, 5(2), 11; https://doi.org/10.3390/proteomes5020011 - 07 Apr 2017
Cited by 35 | Viewed by 8338
Abstract
Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single [...] Read more.
Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single ‘spots’ in a polyacrylamide gel, allowing the quantitation of changes in a proteoform′s abundance to ascertain changes in an organism′s phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the ‘Top-Down’. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O’Farrell’s paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism′s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer. Full article
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1305 KiB  
Review
A Proteomic Perspective on the Bacterial Adaptation to Cold: Integrating OMICs Data of the Psychrotrophic Bacterium Exiguobacterium antarcticum B7
by Rafael A. Baraúna, Dhara Y. Freitas, Juliana C. Pinheiro, Adriana R. C. Folador and Artur Silva
Proteomes 2017, 5(1), 9; https://doi.org/10.3390/proteomes5010009 - 23 Feb 2017
Cited by 28 | Viewed by 6765
Abstract
Since the publication of one of the first studies using 2D gel electrophoresis by Patrick H. O’Farrell in 1975, several other studies have used that method to evaluate cellular responses to different physicochemical variations. In environmental microbiology, bacterial adaptation to cold environments is [...] Read more.
Since the publication of one of the first studies using 2D gel electrophoresis by Patrick H. O’Farrell in 1975, several other studies have used that method to evaluate cellular responses to different physicochemical variations. In environmental microbiology, bacterial adaptation to cold environments is a “hot topic” because of its application in biotechnological processes. As in other fields, gel-based and gel-free proteomic methods have been used to determine the molecular mechanisms of adaptation to cold of several psychrotrophic and psychrophilic bacterial species. In this review, we aim to describe and discuss these main molecular mechanisms of cold adaptation, referencing proteomic studies that have made significant contributions to our current knowledge in the area. Furthermore, we use Exiguobacterium antarcticum B7 as a model organism to present the importance of integrating genomic, transcriptomic, and proteomic data. This species has been isolated in Antarctica and previously studied at all three omic levels. The integration of these data permitted more robust conclusions about the mechanisms of bacterial adaptation to cold. Full article
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1650 KiB  
Review
Isoelectric Point Separations of Peptides and Proteins
by Melissa R. Pergande and Stephanie M. Cologna
Proteomes 2017, 5(1), 4; https://doi.org/10.3390/proteomes5010004 - 25 Jan 2017
Cited by 65 | Viewed by 18399
Abstract
The separation of ampholytic components according to isoelectric point has played an important role in isolating, reducing complexity and improving peptide and protein detection. This brief review outlines the basics of isoelectric focusing, including a summary of the historical achievements and considerations in [...] Read more.
The separation of ampholytic components according to isoelectric point has played an important role in isolating, reducing complexity and improving peptide and protein detection. This brief review outlines the basics of isoelectric focusing, including a summary of the historical achievements and considerations in experimental design. Derivative methodologies of isoelectric focusing are also discussed including common detection methods used. Applications in a variety of fields using isoelectric point based separations are provided as well as an outlook on the field for future studies. Full article
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5670 KiB  
Review
Let There Be Light!
by Doroteya Raykova, Björn Koos, Anna Asplund, Márton Gelléri, Ylva Ivarsson, U. Helena Danielson and Ola Söderberg
Proteomes 2016, 4(4), 36; https://doi.org/10.3390/proteomes4040036 - 29 Nov 2016
Cited by 15 | Viewed by 6620
Abstract
The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Förster resonance energy transfer (FRET) and in situ proximity [...] Read more.
The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Förster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways. Multiple approaches are available for functional analyses in single cells. They provide information not only on the localization of proteins at a given time point, but also on their expression levels and activity states, allowing us to pinpoint hallmarks of different cellular identities within tissues in health and disease. Clever solutions to increase the sensitivity of molecular tools, the possibilities for multiplexing, as well as image resolution have recently been introduced; however, these methods have their pros and cons. Therefore, one needs to carefully consider the biological question of interest along with the nature of the sample before choosing the most suitable method or combination of methods. Herein, we review a few of the most exciting microscopy-based molecular techniques for proteomic analysis and cover the benefits as well as the disadvantages of their use. Full article
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3600 KiB  
Review
Towards the Full Realization of 2DE Power
by Stanislav Naryzhny
Proteomes 2016, 4(4), 33; https://doi.org/10.3390/proteomes4040033 - 15 Nov 2016
Cited by 16 | Viewed by 6515
Abstract
Here, approaches that allow disclosure of the information hidden inside and outside of two-dimensional gel electrophoresis (2DE) are described. Experimental identification methods, such as mass spectrometry of high resolution and sensitivity (MALDI-TOF MS and ESI LC-MS/MS) and immunodetection (Western and Far-Western) in combination [...] Read more.
Here, approaches that allow disclosure of the information hidden inside and outside of two-dimensional gel electrophoresis (2DE) are described. Experimental identification methods, such as mass spectrometry of high resolution and sensitivity (MALDI-TOF MS and ESI LC-MS/MS) and immunodetection (Western and Far-Western) in combination with bioinformatics (collection of all information about proteoforms), move 2DE to the next level of power. The integration of these technologies will promote 2DE as a powerful methodology of proteomics technology. Full article
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2427 KiB  
Review
Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis
by Sandra Murphy, Paul Dowling and Kay Ohlendieck
Proteomes 2016, 4(3), 27; https://doi.org/10.3390/proteomes4030027 - 09 Sep 2016
Cited by 33 | Viewed by 12096
Abstract
The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a [...] Read more.
The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins. Full article
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Other

637 KiB  
Opinion
Top-Down Proteomics and Farm Animal and Aquatic Sciences
by Alexandre M.O. Campos and André M. De Almeida
Proteomes 2016, 4(4), 38; https://doi.org/10.3390/proteomes4040038 - 21 Dec 2016
Cited by 11 | Viewed by 5172
Abstract
Proteomics is a field of growing importance in animal and aquatic sciences. Similar to other proteomic approaches, top-down proteomics is slowly making its way within the vast array of proteomic approaches that researchers have access to. This opinion and mini-review article is dedicated [...] Read more.
Proteomics is a field of growing importance in animal and aquatic sciences. Similar to other proteomic approaches, top-down proteomics is slowly making its way within the vast array of proteomic approaches that researchers have access to. This opinion and mini-review article is dedicated to top-down proteomics and how its use can be of importance to animal and aquatic sciences. Herein, we include an overview of the principles of top-down proteomics and how it differs regarding other more commonly used proteomic methods, especially bottom-up proteomics. In addition, we provide relevant sections on how the approach was or can be used as a research tool and conclude with our opinions of future use in animal and aquatic sciences. Full article
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