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

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

Deadline for manuscript submissions: closed (30 June 2019) | Viewed by 65669

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Special Issue Editors

Prof. Dr. Jens R. Coorssen

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Guest Editor

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
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Dr. Matthew P. Padula

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Guest Editor

School of Life Sciences and Proteomics Core Facility, Faculty of Science, The University of Technology Sydney, Ultimo 2007, Australia
Interests: high-resolution discovery proteomics; methodology development; top-down proteomics; two-dimensional gel electrophoresis; 2DE/MS/MS; proteoforms; lipidomics; metabolomics; data-independent acquisition
Special Issues, Collections and Topics in MDPI journals

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 submissions that pass pre-check are 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 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

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

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Approaches to Top-Down Proteomics: In Honour of Prof. Patrick H. O'Farrell in Proteomes (12 articles)

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Editorial

Jump to: Research, Review, Other

3 pages, 167 KiB

Open AccessEditorial

Special Issue “Top-down Proteomics: In Memory of Dr Alfred Yergey”. Alfred Linwood Yergey, III, 17 September 1941–27 May 2018

by Jens R. Coorssen and Matthew P. Padula

Proteomes 2020, 8(1), 1; https://doi.org/10.3390/proteomes8010001 - 15 Jan 2020

Cited by 1 | Viewed by 2595

Abstract

Please see J [...] Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

Research

Jump to: Editorial, Review, Other

12 pages, 2982 KiB

Open AccessArticle

Targeting a Subset of the Membrane Proteome for Top–Down Mass Spectrometry: Introducing the Proteolipidome

by Julian Whitelegge

Proteomes 2020, 8(1), 5; https://doi.org/10.3390/proteomes8010005 - 10 Mar 2020

Cited by 2 | Viewed by 5139

Abstract

A subsection of integral membrane proteins partition into chloroform during a chloroform/methanol/water extraction primarily designed to extract lipids. Traditionally, these proteins were called proteolipids due to their lipid-like properties; the c-subunit of the ATP synthase integral FO component is the best known due to its abundance. In this manuscript, we investigate purification of proteolipid proteins away from lipids for high-resolution mass spectrometry. Size-exclusion chromatography on silica beads using a chloroform/methanol/aqueous formic acid (4/4/1; v/v) mobile phase allowed the separation of larger proteins (>3 kDa) from lipids (<1.5 kDa) and analysis by online electrospray ionization mass spectrometry. Fraction collection for mass spectrometry was limited by presence of plasticizers and other contaminants solubilized by chloroform. Drying down of the protein sample followed by resuspension in formic acid (70%) allowed reverse-phase chromatography on a polymeric support at elevated temperature, as described previously. Fractions collected in this way could be stored for extended periods at −80 °C without adducts or contaminants. Top–down mass spectrometry enabled the definition of PsaI as a novel proteolipid of spinach thylakoid membrane. Proteolipid preparation worked similarly when total membranes from mouse brains were extracted with chloroform. While it might be tempting to use the described extraction, we prefer to broaden the meaning of the term, whereby the proteolipidome is defined as a novel biological membrane proteome that includes the full complement of membrane proteins, their binding partners/ligands and their tightly bound structural lipids that constitute each protein–lipid complex’s functional unit; that is, a complete description of a biological membrane. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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23 pages, 4157 KiB

Open AccessArticle

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion

by Kendra L. Furber, Peter S. Backlund, Alfred L. Yergey and Jens R. Coorssen

Proteomes 2019, 7(4), 34; https://doi.org/10.3390/proteomes7040034 - 27 Sep 2019

Cited by 5 | Viewed by 3754

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. For 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, whereas high doses inhibited Ca²⁺ sensitivity and extent of vesicle 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. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps 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. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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34 pages, 7732 KiB

Open AccessArticle

Top-Down Proteomics of Medicinal Cannabis

by Delphine Vincent, Steve Binos, Simone Rochfort and German Spangenberg

Proteomes 2019, 7(4), 33; https://doi.org/10.3390/proteomes7040033 - 24 Sep 2019

Cited by 9 | Viewed by 5549

Abstract

The revised legislation on medicinal cannabis has triggered a surge of research studies in this space. Yet, cannabis proteomics is lagging. In a previous study, we optimised the protein extraction of mature buds for bottom-up proteomics. In this follow-up study, we developed a top-down mass spectrometry (MS) proteomics strategy to identify intact denatured protein from cannabis apical buds. After testing different source-induced dissociation (SID), collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) parameters on infused known protein standards, we devised three LC-MS/MS methods for top-down sequencing of cannabis proteins. Different MS/MS modes produced distinct spectra, albeit greatly overlapping between SID, CID, and HCD. The number of fragments increased with the energy applied; however, this did not necessarily translate into greater sequence coverage. Some precursors were more amenable to fragmentation than others. Sequence coverage decreased as the mass of the protein increased. Combining all MS/MS data maximised amino acid (AA) sequence coverage, achieving 73% for myoglobin. In this experiment, most cannabis proteins were smaller than 30 kD. A total of 46 cannabis proteins were identified with 136 proteoforms bearing different post-translational modifications (PTMs), including the excision of N-terminal M, the N-terminal acetylation, methylation, and acetylation of K resides, and phosphorylation. Most identified proteins are involved in photosynthesis, translation, and ATP production. Only one protein belongs to the phytocannabinoid biosynthesis, olivetolic acid cyclase. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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11 pages, 1426 KiB

Open AccessArticle

In Silico Identification of Antimicrobial Peptides in the Proteomes of Goat and Sheep Milk and Feta Cheese

by Marios Tomazou, Anastasis Oulas, Athanasios K. Anagnostopoulos, George Th. Tsangaris and George M. Spyrou

Proteomes 2019, 7(4), 32; https://doi.org/10.3390/proteomes7040032 - 21 Sep 2019

Cited by 15 | Viewed by 5750

Abstract

Milk and dairy products are a major functional food group of growing scientific and commercial interest due to their nutritional value and bioactive “load”. A major fraction of the latter is attributed to milk’s rich protein content and its biofunctional peptides that occur naturally during digestion. On the basis of the identified proteome datasets of milk whey from sheep and goat breeds in Greece and feta cheese obtained during previous work, we applied an in silico workflow to predict and characterise the antimicrobial peptide content of these proteomes. We utilised existing tools for predicting peptide sequences with antimicrobial traits complemented by in silico protein cleavage modelling to identify frequently occurring antimicrobial peptides (AMPs) in the gastrointestinal (GI) tract in humans. The peptides of interest were finally assessed for their stability with respect to their susceptibility to cleavage by endogenous proteases expressed along the intestinal part of the GI tract and ranked with respect to both their antimicrobial and stability scores. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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14 pages, 1480 KiB

Open AccessArticle

Towards Understanding Non-Infectious Growth-Rate Retardation in Growing Pigs

by Ana M. Gutiérrez, Juan Sotillo, Sarah Schlosser, Karin Hummel and Ingrid Miller

Proteomes 2019, 7(3), 31; https://doi.org/10.3390/proteomes7030031 - 11 Sep 2019

Cited by 11 | Viewed by 3583

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 or 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 show any alteration in the saliva of animals with growth-rate retardation, so other pathways should be affected. The present study investigates saliva samples from animals with the same commercial crossbreed, sex and age, comparing control pigs and pigs with growth-rate retardation. A proteomics approach based on two-dimensional gel electrophoresis including mass spectrometry together with validation experiments was applied for the search of proteins that could help understand disease mechanisms and be used for early disease detection. Two proteins were detected as possible markers of growth-rate retardation, specifically S100A12 and carbonic anhydrase VI. A decrease in innate immune response was confirmed in pigs with growth-rate retardation, however further studies should be necessary to understand the role of the different CA VI proteoforms observed. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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20 pages, 5257 KiB

Open AccessArticle

A Proteomic View of Cellular Responses to Anticancer Quinoline-Copper Complexes

by Bastien Dalzon, Joanna Bons, Hélène Diemer, Véronique Collin-Faure, Caroline Marie-Desvergne, Muriel Dubosson, Sarah Cianferani, Christine Carapito and Thierry Rabilloud

Proteomes 2019, 7(2), 26; https://doi.org/10.3390/proteomes7020026 - 24 Jun 2019

Cited by 12 | Viewed by 4861

Abstract

Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a classical proteomic approach based on two-dimensional elelctrophoresis to investigate the mechanisms of action of a hydroxyquinoline-copper complex, which shows promising anticancer activities, using the leukemic cell line RAW264.7 as the biological target. Pathway analysis of the modulated proteins highlighted changes in the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, and carbon metabolism or oxido-reduction. In line with these prteomic-derived hypotheses, targeted validation experiments showed that the hydroxyquinoline-copper complex induces a massive reduction in free glutathione and a strong alteration in the actin cytoskeleton, suggesting a multi-target action of the hydroxyquinoline-copper complex on cancer cells. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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Review

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15 pages, 3290 KiB

Open AccessReview

Innovating the Concept and Practice of Two-Dimensional Gel Electrophoresis in the Analysis of Proteomes at the Proteoform Level

by Xianquan Zhan, Biao Li, Xiaohan Zhan, Hartmut Schlüter, Peter R. Jungblut and Jens R. Coorssen

Proteomes 2019, 7(4), 36; https://doi.org/10.3390/proteomes7040036 - 30 Oct 2019

Cited by 59 | Viewed by 7107

Abstract

Two-dimensional gel electrophoresis (2DE) is an important and well-established technical platform enabling extensive top-down proteomic analysis. However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms. It is time to popularize a new concept of 2DE for proteomics. With the development and enrichment of the proteome concept, any given “protein” is now recognized to consist of a series of proteoforms. Thus, it is the proteoform, rather than the canonical protein, that is the basic unit of a proteome, and each proteoform has a specific isoelectric point (pI) and relative mass (M_r). Accordingly, using 2DE, each proteoform can routinely be resolved and arrayed according to its different pI and M_r. Each detectable spot contains multiple proteoforms derived from the same gene, as well as from different genes. Proteoforms derived from the same gene are distributed into different spots in a 2DE pattern. High-resolution 2DE is thus actually an initial level of separation to address proteome complexity and is effectively a pre-fractionation method prior to analysis using mass spectrometry (MS). Furthermore, stable isotope-labeled 2DE coupled with high-sensitivity liquid chromatography-tandem MS (LC-MS/MS) has tremendous potential for the large-scale detection, identification, and quantification of the proteoforms that constitute proteomes. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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19 pages, 715 KiB

Open AccessFeature PaperReview

What is Normalization? The Strategies Employed in Top-Down and Bottom-Up Proteome Analysis Workflows

by Matthew B. O’Rourke, Stephanie E. L. Town, Penelope V. Dalla, Fiona Bicknell, Naomi Koh Belic, Jake P. Violi, Joel R. Steele and Matthew P. Padula

Proteomes 2019, 7(3), 29; https://doi.org/10.3390/proteomes7030029 - 22 Aug 2019

Cited by 25 | Viewed by 12921

Abstract

The accurate quantification of changes in the abundance of proteins is one of the main applications of proteomics. The maintenance of accuracy can be affected by bias and error that can occur at many points in the experimental process, and normalization strategies are crucial to attempt to overcome this bias and return the sample to its regular biological condition, or normal state. Much work has been published on performing normalization on data post-acquisition with many algorithms and statistical processes available. However, there are many other sources of bias that can occur during experimental design and sample handling that are currently unaddressed. This article aims to cast light on the potential sources of bias and where normalization could be applied to return the sample to its normal state. Throughout we suggest solutions where possible but, in some cases, solutions are not available. Thus, we see this article as a starting point for discussion of the definition of and the issues surrounding the concept of normalization as it applies to the proteomic analysis of biological samples. Specifically, we discuss a wide range of different normalization techniques that can occur at each stage of the sample preparation and analysis process. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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23 pages, 7219 KiB

Open AccessReview

Characterization of Contractile Proteins from Skeletal Muscle Using Gel-Based Top-Down Proteomics

by Paul Dowling, Margit Zweyer, Dieter Swandulla and Kay Ohlendieck

Proteomes 2019, 7(2), 25; https://doi.org/10.3390/proteomes7020025 - 20 Jun 2019

Cited by 26 | Viewed by 9672

Abstract

The mass spectrometric analysis of skeletal muscle proteins has used both peptide-centric and protein-focused approaches. The term ‘top-down proteomics’ is often used in relation to studying purified proteoforms and their post-translational modifications. Two-dimensional gel electrophoresis, in combination with peptide generation for the identification and characterization of intact proteoforms being present in two-dimensional spots, plays a critical role in specific applications of top-down proteomics. A decisive bioanalytical advantage of gel-based and top-down approaches is the initial bioanalytical focus on intact proteins, which usually enables the swift identification and detailed characterisation of specific proteoforms. In this review, we describe the usage of two-dimensional gel electrophoretic top-down proteomics and related approaches for the systematic analysis of key components of the contractile apparatus, with a special focus on myosin heavy and light chains and their associated regulatory proteins. The detailed biochemical analysis of proteins belonging to the thick and thin skeletal muscle filaments has decisively improved our biochemical understanding of structure-function relationships within the contractile apparatus. Gel-based and top-down proteomics has clearly established a variety of slow and fast isoforms of myosin, troponin and tropomyosin as excellent markers of fibre type specification and dynamic muscle transition processes. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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