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Analysis of Peptides and Proteins by Electrophoretic Techniques

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Analytical Chemistry".

Deadline for manuscript submissions: closed (30 April 2019) | Viewed by 29268

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Special Issue Editors

Prof. Dr. Angela R. Piergiovanni

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Guest Editor

Istituto di Bioscienze e BioRisorse, Consiglio Nazionale delle Ricerche (IBBR-CNR), 70126 Bari, Italy
Interests: characterization and evaluation of Italian landraces of legume species; evaluation of grain nutritional quality; measurement of anti-nutritional and toxic compounds; characterization of germplasm segments
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. José Manuel Herrero-Martínez

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Guest Editor

Department of Analytical Chemistry, Faculty of Chemistry, University of Valencia, Burjassot, Spain
Interests: porous materials; nanomaterials; molecular recognition sorbents; sample treatment; solid-phase extraction; capillary electromigration techniques; capillary/nano liquid chromatography; food analysis
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The characterization of real complex matrices containing peptides and proteins constitutes a relevant issue in the research of life and biological sciences. To understand their functions in animals, plants, and food, studies of their expression levels, post-translational modifications, and specific interactions are crucial. Moreover, proteins and peptides are important biomarkers of many human diseases. Increase in our knowledge on this subject could help to develop biopharmaceuticals. Separation techniques, based on electromigration, coupled to sensitive detection methodologies, have enabled researchers to address these challenging issues. Recently, miniaturized electromigration techniques, including separations achieved in narrow fused silica capillaries or other microdevices, have increased separation efficiency and speed of analysis, reducing sample consumption and waste production.

The present Special Issue, “Analysis of Peptides and Proteins by Electrophoretic Techniques”, aims to collect and to spread some of the most significant and recent advancements in the study of theses macromolecules. The scope is broad, regarding the use of capillary electrophoresis (CE), chip-based and other miniaturized supports, as well as slab gels to characterize complex mixtures of peptides and proteins. Relevant applications to real matrices (biological fluids, plant extracts, food, etc.) and new technological developments such as microfluidics hyphenated with mass spectrometry, as well as future directions of these techniques, are welcomed.

Prof. Dr. Angela R. Piergiovanni
Prof. Dr. José Manuel Herrero-Martínez
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. Molecules is an international peer-reviewed open access semimonthly 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 2700 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

slab-gel electrophoresis
capillary electromigration techniques
chip based and capillary based CE
protein and/or peptides mixtures characterization
hyphenation with mass spectrometry
proteomics
metabolomics

Published Papers (6 papers)

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Research

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14 pages, 1472 KiB

Open AccessArticle

Carbon Dot-Mediated Capillary Electrophoresis Separations of Metallated and Demetallated Forms of Transferrin Protein

by Leona R. Sirkisoon, Honest C. Makamba, Shingo Saito and Christa L. Colyer

Molecules 2019, 24(10), 1916; https://doi.org/10.3390/molecules24101916 - 18 May 2019

Cited by 12 | Viewed by 2919

Abstract

Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a ‘bottom-up’ method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis. Full article

(This article belongs to the Special Issue Analysis of Peptides and Proteins by Electrophoretic Techniques)

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10 pages, 1320 KiB

Open AccessArticle

Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE

by Lei Yu, Yonghong Li, Lei Tao, Chuncui Jia, Wenrong Yao, Chunming Rao and Junzhi Wang

Molecules 2019, 24(7), 1210; https://doi.org/10.3390/molecules24071210 - 28 Mar 2019

Cited by 3 | Viewed by 3869

Abstract

During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261–306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12. Full article

(This article belongs to the Special Issue Analysis of Peptides and Proteins by Electrophoretic Techniques)

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11 pages, 1271 KiB

Open AccessArticle

A Simple Method for On-Gel Detection of Myrosinase Activity

by Sándor Gonda, Zsolt Szűcs, Tamás Plaszkó, Zoltán Cziáky, Attila Kiss-Szikszai, Gábor Vasas and Márta M-Hamvas

Molecules 2018, 23(9), 2204; https://doi.org/10.3390/molecules23092204 - 31 Aug 2018

Cited by 6 | Viewed by 4085

Abstract

Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO₄⁻ is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031–0.25 U (pure Sinapis alba myrosinase, R² = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification. Full article

(This article belongs to the Special Issue Analysis of Peptides and Proteins by Electrophoretic Techniques)

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10 pages, 913 KiB

Open AccessCommunication

First Look at the Venom of Naja ashei

by Konrad Kamil Hus, Justyna Buczkowicz, Vladimír Petrilla, Monika Petrillová, Andrzej Łyskowski, Jaroslav Legáth and Aleksandra Bocian

Molecules 2018, 23(3), 609; https://doi.org/10.3390/molecules23030609 - 08 Mar 2018

Cited by 28 | Viewed by 5671

Abstract

Naja ashei is an African spitting cobra species closely related to N. mossambica and N. nigricollis. It is known that the venom of N. ashei, like that of other African spitting cobras, mainly has cytotoxic effects, however data about its specific protein composition are not yet available. Thus, an attempt was made to determine the venom proteome of N. ashei with the use of 2-D electrophoresis and MALDI ToF/ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry techniques. Our investigation revealed that the main components of analysed venom are 3FTxs (Three-Finger Toxins) and PLA₂s (Phospholipases A₂). Additionally the presence of cysteine-rich venom proteins, 5′-nucleotidase and metalloproteinases has also been confirmed. The most interesting fact derived from this study is that the venom of N. ashei includes proteins not described previously in other African spitting cobras—cobra venom factor and venom nerve growth factor. To our knowledge, there are currently no other reports concerning this venom composition and we believe that our results will significantly increase interest in research of this species. Full article

(This article belongs to the Special Issue Analysis of Peptides and Proteins by Electrophoretic Techniques)

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Review

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25 pages, 1020 KiB

Open AccessReview

Application of Capillary Electrophoresis with Laser-Induced Fluorescence to Immunoassays and Enzyme Assays

by Binh Thanh Nguyen and Min-Jung Kang

Molecules 2019, 24(10), 1977; https://doi.org/10.3390/molecules24101977 - 22 May 2019

Cited by 18 | Viewed by 5335

Abstract

Capillary electrophoresis using laser-induced fluorescence detection (CE-LIF) is one of the most sensitive separation tools among electrical separation methods. The use of CE-LIF in immunoassays and enzyme assays has gained a reputation in recent years for its high detection sensitivity, short analysis time, and accurate quantification. Immunoassays are bioassay platforms that rely on binding reactions between an antigen (analyte) and a specific antibody. Enzyme assays measure enzymatic activity through quantitative analysis of substrates and products by the reaction of enzymes in purified enzyme or cell systems. These two category analyses play an important role in the context of biopharmaceutical analysis, clinical therapy, drug discovery, and diagnosis analysis. This review discusses the expanding portfolio of immune and enzyme assays using CE-LIF and focuses on the advantages and disadvantages of these methods over the ten years of existing technology since 2008. Full article

(This article belongs to the Special Issue Analysis of Peptides and Proteins by Electrophoretic Techniques)

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28 pages, 1250 KiB

Open AccessReview

Advances in the Biology of Seed and Vegetative Storage Proteins Based on Two-Dimensional Electrophoresis Coupled to Mass Spectrometry

by Daniel Mouzo, Javier Bernal, María López-Pedrouso, Daniel Franco and Carlos Zapata

Molecules 2018, 23(10), 2462; https://doi.org/10.3390/molecules23102462 - 26 Sep 2018

Cited by 26 | Viewed by 6639

Abstract

Seed storage proteins play a fundamental role in plant reproduction and human nutrition. They accumulate during seed development as reserve material for germination and seedling growth and are a major source of dietary protein for human consumption. Storage proteins encompass multiple isoforms encoded by multi-gene families that undergo abundant glycosylations and phosphorylations. Two-dimensional electrophoresis (2-DE) is a proteomic tool especially suitable for the characterization of storage proteins because of their peculiar characteristics. In particular, storage proteins are soluble multimeric proteins highly represented in the seed proteome that contain polypeptides of molecular mass between 10 and 130 kDa. In addition, high-resolution profiles can be achieved by applying targeted 2-DE protocols. 2-DE coupled with mass spectrometry (MS) has traditionally been the methodology of choice in numerous studies on the biology of storage proteins in a wide diversity of plants. 2-DE-based reference maps have decisively contributed to the current state of our knowledge about storage proteins in multiple key aspects, including identification of isoforms and quantification of their relative abundance, identification of phosphorylated isoforms and assessment of their phosphorylation status, and dynamic changes of isoforms during seed development and germination both qualitatively and quantitatively. These advances have translated into relevant information about meaningful traits in seed breeding such as protein quality, longevity, gluten and allergen content, stress response and antifungal, antibacterial, and insect susceptibility. This review addresses progress on the biology of storage proteins and application areas in seed breeding using 2-DE-based maps. Full article

(This article belongs to the Special Issue Analysis of Peptides and Proteins by Electrophoretic Techniques)

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