Special Issue "Top-down Proteomics: In Memory of Dr. Alfred Yergey"

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

Deadline for manuscript submissions: 30 June 2019

Special Issue Editors

Guest Editor
Prof. Jens R. Coorssen

Department of Health Sciences, Faculty of Applied Health Sciences, and Department of Biological Sciences, Faculty of Mathematics and Science, Brock University, ON, Canada
Website | E-Mail
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
Guest Editor
Dr. Matthew P. Padula

Proteomics Core Facility and School of Life Sciences, University of Technology Sydney, Ultimo, NSW, Australia
Website | E-Mail
Interests: high-resolution discovery proteomics; methodology development; top-down proteomics; two-dimensional gel electrophoresis; 2DE/MS/MS; proteoforms; lipidomics; metabolomics; data-independent acquisition

Special Issue Information

Dear Colleagues,

The legacy of Al Yergey is the belief that if you are going to do something, make sure that you do it properly and quantitatively—a sentiment best conveyed by his article “Proteomics Is Analytical Chemistry: Fitness-for-Purpose in the Application of Top-Down and Bottom-Up Analyses” in which he appealed to the field of proteomics to hold itself to the highest possible standard. In this Special Issue, we want to further explore that ideal by publishing articles and reviews that demonstrate the state-of-the-art in top-down proteomics and complementarity with bottom-up approaches. Emphasis will be on the absolute need for analytical rigor and reproducibility. We thus seek to have a conversation about how top-down proteomics—indeed, proteomics as a whole—needs to progress to best serve analytical science, and the ever-growing role of proteomics as a cornerstone of modern biological/biomedical research. Simply put, proteomes are made up of proteoforms rather than simply amino acid sequences or open reading frames. We must be cognizant of this sheer molecular reality in terms of molecular mechanisms and the identification of effective biomarkers and therapeutic targets. Do our most current and most widely touted approaches really capture, let alone address, this reality? How do we find a more collegial, collaborative, and complementary way forward? In the spirit of Al Yergey’s substantial legacy as both a researcher and genuine Renaissance man (his main hobbies were formal botanical drawing and brewing beer!), we hope this Special Issue can highlight more productive ways forward than the field has perhaps been entertaining for the last decade or so. This comes with the recognition that no approach is “perfect”, but that by not recognizing the genuine pros and cons of the available analytical approaches (which has not largely been the case with the preponderance of dogma in the field) we cannot move effectively forward in the most productive collaborative manner.

Prof. Jens R. Coorssen
Dr. Matthew P. Padula
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Proteomes is an international peer-reviewed open access quarterly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 550 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.


  • analytical proteomics
  • biomarkers
  • bottom-up proteomics
  • chromatography
  • computational analysis
  • electrophoresis
  • molecular mechanisms
  • protein species/proteoforms
  • mass spectrometry (LC/MS/MS)
  • top-down proteomics
  • post-translational modifications
  • two-dimensional gel electrophoresis

Related Special Issue

Published Papers

This special issue is now open for submission, see below for planned papers.

Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Title: Characterisation of contractile proteins from skeletal muscle using two-dimensional gel electrophoretic top-down proteomics
Authors: Kay Ohlendieck, Paul Dowling et al.
Affiliation: Department of Biology, Maynooth University, National University of Ireland Maynooth, Co. Kildare, Ireland
Email: [email protected]; [email protected]
Abstract: The contractile function of the skeletal muscle system is based on the dynamic arrangement of an unusual cell fibre type that is very large, multi-nucleated and extremely heterogeneous in its protein isoform composition. Over the last decade, both peptide-centric and protein-focused approaches were used for the systematic mass spectrometry-based proteomic analysis of skeletal muscle proteins. The application of top-down proteomics (TDP) provided data for absolute protein mass determination and detailed information on the identity of specific protein isoforms and their post-translational modifications. This makes TDP an ideal investigative tool of muscle protein biochemistry for determining the structure and function of macromolecular protein complexes. This review gives a general outline of the TDP approach in relation to skeletal muscle biology and focuses especially on the application of two-dimensional gel electrophoresis for studying alterations in distinct proteoforms of contractile proteins during physiological adaptations versus pathophysiological degeneration in the skeletal musculature.

Title: A proteomic view of cellular responses to anticancer quinoline-copper complexes
Author: Thierry Rabilloud
Affiliation: University Grenoble Alpes, Chemistry and Biology of Metals, CEA BIG, CNRS UMR 5249, F-38054 Grenoble, France
Email: [email protected]
Abstract: Copper chelates have been shown to exhibit interesting anticancer properties, bit their mechanism of action is still unclear. Proteasome inhibition is the main suggested mechanism, but other mechanism targeting histone deacetylase or the mTOR pathway. To get a better appraisal of the effects of copper chelates on cancer cells, we used a 2D gel-based proteomics approach. This approach detected the uniquitin-proteasome pathway as altered, but highlighted also several other pathways and putative targets, such as the endosomal-lysosomal pathway, the mitochondria or the actin cytoskeleton. Targeted validation experiments confirmed alterations in these pathways upon exposure of cancer cells to copper chelates, even at subtoxic doses.

Title: Top-down proteomics on chromatographic time scales using a using a novel ECD-QTOF configuration
Author: Blaine R. Roberts
Affiliation: The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, Australia
Email: [email protected]
Abstract: One limitation of a bottom-up proteomic strategy, which first digests proteins into peptides, is the lack of ability to determine the number of proteoforms present. Although bottom-up proteomics can described many post-translationally modified peptides associated with a single protein, it is not clear what the molar ratio of the PTMs were in the starting intact mixture. In other words, bottom-up provides the building pieces but not the information required to determine the protoforms that were present in the original sample. Top-down proteomics aims to bridge this gap by directly measuring the intact proteoforms. However, the technology required for top-down proteomics is not as mature or readily available as the current bottom-up technology. Here we describe the retrofitting of an QTOF mass spectrometer with an electron capture disassociation (ECD) cell. The ECD cells provides a means to fragment intact proteins. We characterize the use of the this cell on the QTOF and demonstrate the ability to sequence proteins up to ~ 30kDa. The protein spectra consisted primarily of c and z ions, though the ECD cell also produced a substantial number of d and w sidechain fragments. These side-chain fragments allow leucine/isoleucine or lysine/glutamine pairs to be distinguished, facilitating de novo sequencing. Labile post-translational modifications were also retained. The copper and zinc cofactors in superoxide dismutase (17 kDa) remained bound to their respective binding sites in ECD fragments. We then applied this technology to protein extracts from human brain to show that the ECD-QTOF can work on chromatographic time scales, in an automated fashion similar to data dependent acquisition used in bottom-up proteomics, to provide > 80% sequence coverage of proteins extracted from human brain tissue.

Title: Development of a database for inventory of human proteoforms
Author: Stanislav Naryzhny1,2
Affiliations: 1Orekhovich Institute of Biomedical Chemistry of Russian Academy of Medical Sciences, Pogodinskaya 10, Moscow, 119121, Russia. E-mail: [email protected]; 2B.P. Konstantinov Petersburg Nuclear Physics Institute, National Research Center "Kurchatov Institute", Gatchina, Leningrad region, 188300, Russia; Correspondence: [email protected]; Tel.: (+7) 9111764453
Email: [email protected]
Abstract: The main complexity in the human proteome is that it is composed from diverse and heterogeneous gene products/proteoforms. In decoding of this intricacy significant advances by “Top-Down” proteomics have been made. Previously we have been discussing the main technical aspects in developing for inventory of  human proteoforms that would be visually attractive, clear, easy to search and perceptive. Here, we describe a development of our first draft of the database of glioblastoma proteins (proteoforms) that is based on this discussion.

Title: Towards understanding non-infectious growth-rate retardation in growing pigs
Authors: Gutiérrez A.M.1,*, Miller I.2
Affiliations: 1Department of Animal Medicine and Surgery, BioVetMed research group, University of Murcia, Spain; 2Department of Biomedical Sciences, University of Veterinary Medicine of Vienna, Austria
Emails: [email protected]; [email protected]
Abstract: For growth-rate retardation in commercial growing pigs suffering from non-infectious diseases, no biomarker is available for early detection and prevention of the condition nor for the diagnosis of affected animals. The point in question is that the underlying pathological pathway of the condition is still unknown and multiple nutritional or management issues could be the cause of the disease. Common health status markers such as acute phase proteins, adenosine deaminase activity or total antioxidant capacity did not shown any alteration in the saliva of animals with growth-rate retardation, so other pathways seem affected. The present study investigates saliva samples from animals with the same commercial crossbreed, sex and age, comparing healthy pigs and pigs with growth-rate retardation. A proteomics approach based on the combination of two-dimensional gel electrophoresis and mass spectrometry was applied for the search of proteins that could help understand disease mechanisms.

Title: Quantiative Proteomic Analysis in Niemann-Pick Disease, Type C1 Patient Fibroblasts with PI3K Modulators
Authors: Stephanie M. Cologna and Mi Nguyen
Affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, USA
Email: [email protected]
Abstract: Niemann-Pick Disease, Type C1 (NPC1) is a fatal, neurodegenerative disorder characterized by endo-lysosomal accumulation of cholesterol and sphingolipids.  Cerebellar degeneration is a hallmark of the disease as well as enlargement of liver and spleen.  In previous proteomic studies from our group, we identified that enzymes responsible for the biosynthesis of phosphoinositides are altered in multiple NPC1 tissues.  In the current studie, we used control and NPC1 patient fibroblasts to evaluate inhibition and activation of a critical phosphoinositide kinase, namely, PI3K.  Differential proteomics was carried out using TMT-6 isobaric labeling. Changes in the proteome provide insight into a targetable pathway for NPC1 treatment.

Title: Unbiased thiol-labeling approach reveals multiple roles for Rab proteins in regulated secretion
Authors: Kendra L Furber, Peter S Backlund, Alfred L Yergey and Jens R Coorssen
Affiliation: Department of Health Sciences, Faculty of Applied Health Sciences, and Department of Biological Sciences, Faculty of Mathematics and Science, Brock University, ON, Canada
Emails: [email protected]; [email protected]; [email protected]
Abstract: Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. As an unbiased, top-down proteomic approach, select thiol-reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2 sensitivity, but high doses inhibited fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol-reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Two-dimensional gel electrophoresis of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions followed by mass spectrometric analyses identified several candidate targets, some of which have been previously linked to the late stages of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity and thus the efficiency of membrane fusion which may, in part, be linked to their previously identified upstream roles in vesicle docking.

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