Masses and sequences of peptides found in a glycopeptide-enriched fraction from porcine IgG (wild-type) tryptic digestion products. Red: Labeled manually; black: Sequenced and/or matched by GPM [24]. Glycopeptides,
\\n\\n
IntechOpen was founded by scientists, for scientists, in order to make book publishing accessible around the globe. Over the last two decades, this has driven Open Access (OA) book publishing whilst levelling the playing field for global academics. Through our innovative publishing model and the support of the research community, we have now published over 5,700 Open Access books and are visited online by over three million academics every month. These researchers are increasingly working in broad technology-based subjects, driving multidisciplinary academic endeavours into human health, environment, and technology.
\\n\\nBy listening to our community, and in order to serve these rapidly growing areas which lie at the core of IntechOpen's expertise, we are launching a portfolio of Open Science journals:
\\n\\nAll three journals will publish under an Open Access model and embrace Open Science policies to help support the changing needs of academics in these fast-moving research areas. There will be direct links to preprint servers and data repositories, allowing full reproducibility and rapid dissemination of published papers to help accelerate the pace of research. Each journal has renowned Editors in Chief who will work alongside a global Editorial Board, delivering robust single-blind peer review. Supported by our internal editorial teams, this will ensure our authors will receive a quick, user-friendly, and personalised publishing experience.
\\n\\n"By launching our journals portfolio we are introducing new, dedicated homes for interdisciplinary technology-focused researchers to publish their work, whilst embracing Open Science and creating a unique global home for academics to disseminate their work. We are taking a leap toward Open Science continuing and expanding our fundamental commitment to openly sharing scientific research across the world, making it available for the benefit of all." Dr. Sara Uhac, IntechOpen CEO
\\n\\n"Our aim is to promote and create better science for a better world by increasing access to information and the latest scientific developments to all scientists, innovators, entrepreneurs and students and give them the opportunity to learn, observe and contribute to knowledge creation. Open Science promotes a swifter path from research to innovation to produce new products and services." Alex Lazinica, IntechOpen founder
\\n\\nIn conclusion, Natalia Reinic Babic, Head of Journal Publishing and Open Science at IntechOpen adds:
\\n\\n“On behalf of the journal team I’d like to thank all our Editors in Chief, Editorial Boards, internal supporting teams, and our scientific community for their continuous support in making this portfolio a reality - we couldn’t have done it without you! With your support in place, we are confident these journals will become as impactful and successful as our book publishing program and bring us closer to a more open (science) future.”
\\n\\nWe invite you to visit the journals homepage and learn more about the journal’s Editorial Boards, scope and vision as all three journals are now open for submissions.
\\n\\nFeel free to share this news on social media and help us mark this memorable moment!
\\n\\n\\n"}]',published:!0,mainMedia:{caption:"",originalUrl:"/media/original/237"}},components:[{type:"htmlEditorComponent",content:'
After years of being acknowledged as the world's leading publisher of Open Access books, today, we are proud to announce we’ve successfully launched a portfolio of Open Science journals covering rapidly expanding areas of interdisciplinary research.
\n\n\n\nIntechOpen was founded by scientists, for scientists, in order to make book publishing accessible around the globe. Over the last two decades, this has driven Open Access (OA) book publishing whilst levelling the playing field for global academics. Through our innovative publishing model and the support of the research community, we have now published over 5,700 Open Access books and are visited online by over three million academics every month. These researchers are increasingly working in broad technology-based subjects, driving multidisciplinary academic endeavours into human health, environment, and technology.
\n\nBy listening to our community, and in order to serve these rapidly growing areas which lie at the core of IntechOpen's expertise, we are launching a portfolio of Open Science journals:
\n\nAll three journals will publish under an Open Access model and embrace Open Science policies to help support the changing needs of academics in these fast-moving research areas. There will be direct links to preprint servers and data repositories, allowing full reproducibility and rapid dissemination of published papers to help accelerate the pace of research. Each journal has renowned Editors in Chief who will work alongside a global Editorial Board, delivering robust single-blind peer review. Supported by our internal editorial teams, this will ensure our authors will receive a quick, user-friendly, and personalised publishing experience.
\n\n"By launching our journals portfolio we are introducing new, dedicated homes for interdisciplinary technology-focused researchers to publish their work, whilst embracing Open Science and creating a unique global home for academics to disseminate their work. We are taking a leap toward Open Science continuing and expanding our fundamental commitment to openly sharing scientific research across the world, making it available for the benefit of all." Dr. Sara Uhac, IntechOpen CEO
\n\n"Our aim is to promote and create better science for a better world by increasing access to information and the latest scientific developments to all scientists, innovators, entrepreneurs and students and give them the opportunity to learn, observe and contribute to knowledge creation. Open Science promotes a swifter path from research to innovation to produce new products and services." Alex Lazinica, IntechOpen founder
\n\nIn conclusion, Natalia Reinic Babic, Head of Journal Publishing and Open Science at IntechOpen adds:
\n\n“On behalf of the journal team I’d like to thank all our Editors in Chief, Editorial Boards, internal supporting teams, and our scientific community for their continuous support in making this portfolio a reality - we couldn’t have done it without you! With your support in place, we are confident these journals will become as impactful and successful as our book publishing program and bring us closer to a more open (science) future.”
\n\nWe invite you to visit the journals homepage and learn more about the journal’s Editorial Boards, scope and vision as all three journals are now open for submissions.
\n\nFeel free to share this news on social media and help us mark this memorable moment!
\n\n\n'}],latestNews:[{slug:"webinar-introduction-to-open-science-wednesday-18-may-1-pm-cest-20220518",title:"Webinar: Introduction to Open Science | Wednesday 18 May, 1 PM CEST"},{slug:"step-in-the-right-direction-intechopen-launches-a-portfolio-of-open-science-journals-20220414",title:"Step in the Right Direction: IntechOpen Launches a Portfolio of Open Science Journals"},{slug:"let-s-meet-at-london-book-fair-5-7-april-2022-olympia-london-20220321",title:"Let’s meet at London Book Fair, 5-7 April 2022, Olympia London"},{slug:"50-books-published-as-part-of-intechopen-and-knowledge-unlatched-ku-collaboration-20220316",title:"50 Books published as part of IntechOpen and Knowledge Unlatched (KU) Collaboration"},{slug:"intechopen-joins-the-united-nations-sustainable-development-goals-publishers-compact-20221702",title:"IntechOpen joins the United Nations Sustainable Development Goals Publishers Compact"},{slug:"intechopen-signs-exclusive-representation-agreement-with-lsr-libros-servicios-y-representaciones-s-a-de-c-v-20211123",title:"IntechOpen Signs Exclusive Representation Agreement with LSR Libros Servicios y Representaciones S.A. de C.V"},{slug:"intechopen-expands-partnership-with-research4life-20211110",title:"IntechOpen Expands Partnership with Research4Life"},{slug:"introducing-intechopen-book-series-a-new-publishing-format-for-oa-books-20210915",title:"Introducing IntechOpen Book Series - A New Publishing Format for OA Books"}]},book:{item:{type:"book",id:"2985",leadTitle:null,fullTitle:"Smoothing, Filtering and Prediction - Estimating The Past, Present and Future",title:"Smoothing, Filtering and Prediction",subtitle:"Estimating The Past, Present and Future",reviewType:"peer-reviewed",abstract:"This book describes the classical smoothing, filtering and prediction techniques together with some more recently developed embellishments for improving performance within applications. 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Jeong",slug:"jin-su-jeong",email:"jsbliss@gmail.com",position:null,institution:null},{id:"223557",title:"Prof.",name:"Julio",middleName:null,surname:"Hernández-Blanco",fullName:"Julio Hernández-Blanco",slug:"julio-hernandez-blanco",email:"juliohb@unex.es",position:null,institution:null},{id:"223558",title:"Prof.",name:"Lorenzo",middleName:null,surname:"García-Moruno",fullName:"Lorenzo García-Moruno",slug:"lorenzo-garcia-moruno",email:"lgmoruno@unex.es",position:null,institution:null}]},book:{id:"6066",title:"Landscape Architecture",subtitle:"The Sense of Places, Models and Applications",fullTitle:"Landscape Architecture - The Sense of Places, Models and Applications",slug:"landscape-architecture-the-sense-of-places-models-and-applications",publishedDate:"September 19th 2018",bookSignature:"Amjad Almusaed",coverURL:"https://cdn.intechopen.com/books/images_new/6066.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",editors:[{id:"110471",title:"Prof.",name:"Amjad",middleName:"Zaki",surname:"Almusaed",slug:"amjad-almusaed",fullName:"Amjad Almusaed"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}}},ofsBook:{item:{type:"book",id:"11652",leadTitle:null,title:"Thyroid Cancer - The Road From Genes to Successful Treatment",subtitle:null,reviewType:"peer-reviewed",abstract:"
\r\n\tThyroid cancer is the most common endocrine cancer. Its incidence and prevalence are increasing worldwide. Extensive research has completely changed the landscape of the biology, pathophysiology, and treatment of thyroid cancer. It has become evident that some forms of thyroid cancer have a benign biologic behavior, while others have a more malignant one. For progressive thyroid cancer, multiple growth factors and kinase inhibitors may be beneficial. Treatment of thyroid cancer with radioiodine has been also completely revised. For the reader, it is important to know everything about the incidence and prevalence of thyroid cancer. It is also important to know the result of in-depth research on the biology and pathophysiology of thyroid cancer. It is also important to recognize factors relevant to the benign biologic behavior of the disease. The advent of multiple growth factors and kinase inhibitors has revolutionized the management of the disease. The indications and adverse effects of these agents are important to know. The long-term prognosis of the disease is also an important subject and will be addressed in this book.
",isbn:"978-1-80356-285-8",printIsbn:"978-1-80356-284-1",pdfIsbn:"978-1-80356-286-5",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,hash:"b74aed61ed1639fc9ad70c20d591a10c",bookSignature:"Dr. Ifigenia Kostoglou-Athanassiou",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11652.jpg",keywords:"Genes, Mutations, Thyroidectomy, Lymph Nodes, Resection, Treatment, Multikinase Inhibitors, Growth Factor Inhibitors, Molecular Targeted Treatment, Radioiodine Treatment, Side Effects, Prognostic Factors",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"February 11th 2022",dateEndSecondStepPublish:"April 15th 2022",dateEndThirdStepPublish:"June 14th 2022",dateEndFourthStepPublish:"September 2nd 2022",dateEndFifthStepPublish:"November 1st 2022",remainingDaysToSecondStep:"a month",secondStepPassed:!0,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"An active clinical endocrinologist, head of the endocrine department of the Asclepeion Hospital, Athens, awarded her Ph.D. from the Univerisity of London. Dr. obtained her medical degree at the University of Athens and is a member of the European Society of Endocrinology and Hellenic Endocrine Society.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"307495",title:"Dr.",name:"Ifigenia",middleName:null,surname:"Kostoglou-Athanassiou",slug:"ifigenia-kostoglou-athanassiou",fullName:"Ifigenia Kostoglou-Athanassiou",profilePictureURL:"https://mts.intechopen.com/storage/users/307495/images/system/307495.jpg",biography:"Dr. Ifigenia Kostoglou-Athanassiou was born in Thessaloniki, Greece. She is an endocrinologist who graduated from the Medical School, Aristotle University of Thessaloniki, Greece. She obtained an MD from the University of Athens Medical School, and a Ph.D. from the University of London. Her areas of research include breast cancer, neuroendocrinology, melatonin, thyroid cancer, vitamin D, and autoimmune diseases. She has won several awards for her research. Dr. Kostoglou-Athanassiou has published numerous papers and book chapters. Currently, she works as a consultant endocrinologist and head of the Endocrine Department, Asclepeion Hospital, Voula, Athens, Greece.",institutionString:"Department of Endocrinology, Asclepeion Hospital",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"3",totalChapterViews:"0",totalEditedBooks:"1",institution:null}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"444318",firstName:"Nika",lastName:"Karamatic",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/444318/images/20011_n.jpg",email:"nika@intechopen.com",biography:"As an Author Service Manager, my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. Mauricio Barría",coverURL:"https://cdn.intechopen.com/books/images_new/6550.jpg",editedByType:"Edited by",editors:[{id:"88861",title:"Dr.",name:"R. Mauricio",surname:"Barría",slug:"r.-mauricio-barria",fullName:"R. Mauricio Barría"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"9500",title:"Recent Advances in Bone Tumours and Osteoarthritis",subtitle:null,isOpenForSubmission:!1,hash:"ea4ec0d6ee01b88e264178886e3210ed",slug:"recent-advances-in-bone-tumours-and-osteoarthritis",bookSignature:"Hiran Amarasekera",coverURL:"https://cdn.intechopen.com/books/images_new/9500.jpg",editedByType:"Edited by",editors:[{id:"67634",title:"Dr.",name:"Hiran",surname:"Amarasekera",slug:"hiran-amarasekera",fullName:"Hiran Amarasekera"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophile",surname:"Theophanides",slug:"theophile-theophanides",fullName:"Theophile Theophanides"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"65007",title:"Characterization of Whole and Fragmented Wild-Type Porcine IgG",doi:"10.5772/intechopen.82792",slug:"characterization-of-whole-and-fragmented-wild-type-porcine-igg",body:'There have been reports on the mass spectrometric (MS) analysis of pig immunoglobulins (IgG) in relationship with use in a xenotransplantation context [1, 2, 3, 4]. These studies have explored the amino acid composition and glycosylation of pig IgG according to glycoproteomic [2, 3] and glycomic [1] workflows involving the enzymatic digestion of whole antibodies. Glycoproteomic workflows resulted in the identification of many peptides that could be matched with the conserved gamma portion of the heavy chains of some of the 11 subtypes of pig IgG [5], including
For more specificity, the analysis of antibodies by MS can take great advantage of enzymatic fragmentation with papain [9, 10] or new enzymes produced by recombinant methods and available on the market [11]. This type of procedure has not been reported for the fragmentation of porcine IgG, to the authors’ knowledge. For instance, procedures have been published for mouse [12, 13], chicken [14], rabbit [15], sheep [16], and human [17, 18] IgGs. The two groups of antibody fragments of primary interest are the antigen-binding fragments (Fab) and class-defining crystallizable fragments (Fc). The hinge region of immunoglobulins (IgGs) is readily accessible to proteolytic attack by enzymes [9, 10], and cleavage at that point produces F(ab’)2 or Fab fragments and the Fc fragment. Papain is a nonspecific thiol-endopeptidase and has a sulfhydryl group in its active site, which must be reduced for activity. When IgG molecules are incubated with papain in the presence of a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragments and one Fc fragment [9, 10].
Fabulous™ enzyme is a recombinant cysteine protease that under reduced conditions digests in the hinge region of antibodies from many species and subclasses, including human, mouse, rat, and goat, yielding Fab and Fc fragments [11]. As a reducing agent is present during digestion, it is likely that interchain thiols will be reduced. Fabulous™ and papain have typically been used for the production of relatively large amounts of antibody fragments (10 mg of starting material), whereas methods adapted to MS require much less, about 50–200 μg. There is a need for downscaling these workflows, especially for porcine IgG, which has not been previously studied by fragmentation followed by MS.
Reports on post-fragmentation MS analyses of antibodies have demonstrated that detailed fragment characterization allows for the identification of more glycosylation sites than bottom-up approaches [19, 20]. It is also important to study amino acid sequences of the variable portions of IgG for therapeutic purposes [21], and the information obtained after fragmentation is much more specific than data generated through the tryptic digestion of whole intact antibodies.
The interest of the present study is to compare results from two main workflows aimed at characterizing wild-type porcine IgG’s glycosylation and amino acid sequence features. In the first workflow, porcine IgG is digested with trypsin, followed by glycopeptide enrichment. Reversed-phase high-performance liquid chromatography (RPLC) coupled with electrospray ionization (ESI) MS and tandem MS (MS/MS) is used to characterize the glycopeptide-rich fraction. The second workflow involves subjecting porcine IgG to fragmentation by one of two enzymes, papain or Fabulous™. The steps necessary between fragmentation and MS included size-exclusion chromatography (SEC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and tryptic digestion. This is the first attempt to characterize porcine IgGs in small amounts (sub-mg) using a combination of these methods. Two different MS techniques were used for the analysis of tryptic products of antibody fragments: matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and ESI/MS–MS coupled with RPLC.
As the tryptic digestion products of whole wild-type porcine IgG antibodies have been characterized by MALDI-MS [2, 3] and ESI-MS and MS/MS (new results presented in this report), data from these different workflows serve as comparative benchmarks between intact and fragmented IgG samples. Overlaps and differences allow to identify peptides and glycopeptides as originating from either the Fc or Fab portions, and database searches [22] can verify if these sequences are already available in the literature or need to be determined
Wild-type porcine IgGs were obtained from Université de Nantes (Dr. Jean-Paul Soulillou’s Laboratory). Trypsin Ultra™ was purchased from Promega (Wisconsin, USA). The Fabulous™ enzyme was kindly provided by Genovis (Cambridge, MA). Dihydroxybenzoic acid (DHB), sinapinic acid, ammonium bicarbonate, dithiothreitol (DTT), L-cysteine, iodoacetamide (IAA), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO). Mini-Protean TGX precast gels (4–15%), Precision Plus™ protein standard, 2-mercaptoethanol, and 4x Laemmli sample buffer were obtained from Bio-Rad (Hercules, CA). Imperial™ protein stain, tris base (2-amino-2-(hydroxymethyl)-1,3-propanediol) and immobilized papain-cross linked and 6% in beaded agarose supplied as 50% glycerol in sodium acetate pH 4.5 were purchased from Thermo Scientific (Rockford, IL). Strata-X C-18 cartridges were obtained from Phenomenex (Torrance, CA). Acetonitrile (ACN) was purchased from EMD-Millipore (Darmstadt, Germany). Sodium phosphate dibasic anhydrous was purchased from McArthur Chemical Co. Ltd. (Montreal, Canada). Hydrochloric acid was purchased from Anachemia (Vancouver, Canada) and deionized water was obtained from an adapted filtration system (Millipore).
Porcine IgG (200 μg) was dissolved in 100 mM ammonium bicarbonate (pH~8) and vortexed. A DTT solution (10 mM) was added to the sample, which was then vortexed and left to react at 56°C for 40 min, then cooled to room temperature. After 500 mM IA was added, and the sample was left to react in the dark for 45 min. The excess of IA was quenched with the addition of 100 mM DTT, and the sample was left to react for 10 min in the dark. Trypsin was added and proteolysis took place at 37°C for ~18 h. To deactivate trypsin, the sample was frozen and dried under vacuum. Glycopeptide enrichment was then performed using a ProteoExtract™ glycopeptide enrichment kit (Millipore-Sigma, Etobicoke, ON) according to the manufacturer’s procedure [23].
Just before use, 20 mM sodium phosphate digestion buffer was prepared with a 10 mM cysteine content, and the pH was adjusted to 7 Bead-immobilized papain slurry (20 μL, 50%) was added to an Eppendorf™ tube. The beads were washed twice with 160 μL of digestion buffer and then re-suspended in the buffer. Porcine IgG (200 μg) was dissolved in the digestion buffer. This was added to the tube containing the washed immobilized papain and digestion buffer. The sample was incubated for ~24 h at 37°C. Constant mixing of the bead slurry was maintained during the whole incubation. The bead-immobilized enzyme was separated from the digest by centrifugation and 20 μL of 10 mM Tris–HCl, pH 7.5 was added before centrifugation. The supernatant, which contained the IgG fragments, was removed.
The IgG sample (200 μg) was added to 200 units of Fabulous™ enzyme in 200 μL of 10 mM Tris, 50 mM cysteine buffer. The sample was vortexed and incubated at 37°C for 1 h.
The digestion mixtures were injected into a preconditioned SEC-300 4.6 × 300 mm silica-based column (Phenomenex, Torrance, CA). The mixtures were eluted with a mobile phase of 0.1% TFA, 0.1% formic acid in 20% ACN at a flow rate of 0.3 mL/min (manufacturer’s recommendation). The HPLC system used was a Waters 1525 binary pump equipped with a Waters 2707 autosampler and a Waters 2998 photodiode array detector (Milford, MA). Fractions were collected, dried, and re-suspended for MALDI-MS analysis.
Once fractionated by SEC, Fab and Fc components were separated on a Mini-Protean™ Tetra cell system (Bio-Rad). Bio-Rad TGX™ gels used had 10 wells and a density gradient of 4–15%. Wells were washed individually four times with running buffer (10 × tris-glycine-SDS buffer diluted 1 × with water) prior to the loading samples. Each sample fraction containing Fab, Fc, or both Fab and Fc had its own lane on the gel. Each gel was loaded with 15 μL of each fraction (in water) in 11.3 μL of 4 × Laemmli sample buffer, without adding 2-mercaptoethanol. Well 1 was loaded with 10 μL of Precision Plus Protein Kaleidoscope™ standard. Intact-reduced IgG (15 μL, ~14 μg) was loaded into well 2. IgG fragments (~14 μg) were loaded in other lanes. Running buffer was poured in the cell system and the voltage was set at 150 V. Samples were allowed to migrate for 40 min, until the dye front reached the bottom of the gel. The gel was removed from the cell and was rinsed four times with Millipore water, and sufficient Imperial™ protein stain was added. IgG fragments absorbed the stain overnight, and the stain was decanted and replaced with Millipore water until gel bands became visible.
Non-reduced Fab and Fc bands were excised from the gel. Tryptic digestion was performed on each single cut out band. Bands were cut into small pieces and placed into 1.5 mL Eppendorf tubes. The digestion buffer was 50 mM ammonium bicarbonate in water. Each tube contained one lane worth of gel. Gel pieces were washed with wash buffer (1:1 digestion buffer-ethanol) until all protein stain was removed. They were then incubated in absolute ethanol for 10 min. Gel pieces were then rewashed with digestion buffer for 20 min and then incubated again in absolute ethanol for 20 min, which was removed from the gel by vacuum centrifugation. Trypsin solution was added and the tubes were placed on ice where the gel was allowed to swell. Thereafter, excess trypsin solution was discarded. Gel pieces were covered with digestion buffer and incubated at 37°C for ~18 h. Glycopeptides and peptides were extracted on C18 cartridges with buffer A, 0.5% acetic acid; extraction buffer B, 5:3 30%; ACN, 0.5% acetic acid; and extraction buffer C, 100% ACN. Samples were dried down for further analysis.
A solution of PNGase F (4 μL, 10 units/μL) was added to a solution of tryptic glycopeptides. The sample was vortexed and set at 37°C for ~18 h. After the digest, glycans were separated from the de-glycosylated peptides on a C18 cartridge. The cartridge was conditioned with 5 x 1 mL (ACN + 0.1% TFA), then 5 × 1 mL of (H2O + 0.1% TFA). The sample was loaded onto the cartridge and allowed to equilibrate for about 1–2 min. Glycans were eluted with 3 mL H2O + 0.1% TFA and collected in two fractions. Peptides were eluted with 1.5 mL of 50:50 ACN:H2O + 0.1% TFA. Solvent was evaporated from the fractions.
Glycopeptide fractions were mixed directly with 5 μL of DHB in 30:70 ACN: water. Samples (1 μL) were then spotted onto the stainless steel target and allowed to dry, for reflector positive mode MALDI-MS. For the Fab and Fc fragments from HPLC fractions, 5 μL of 0.1% TFA and 5 μL of sinapinic acid in 0.1% TFA, 30:70 ACN: water was added, and 1 μL was spotted onto the target already pre-spotted with 0.5 μL of sinapinic acid in ethanol. Spots were then allowed to dry for linear positive mode MALDI-MS.
These analyses were performed on an UltraFleXtreme™ mass spectrometer (Bruker, Billerica, MA) equipped with LID-LIFT™ technology for tandem MS experiments. For positive ionization mode, a nine-peptide calibration mixture with masses ranging from 500 to 5000 Da was used. In linear positive mode, the instrument was calibrated using tryptic peptides of cytochrome C, mass ranging from 10,000 Da to 160,000 Da.
For the Fab and Fc tryptic digest fragments, 100 μL of water was added to the pooled C18 cartridge fractions of each Fab and Fc tryptic digests. Samples were sonicated and then ready for HPLC–MS analysis.
Both digestion mixtures (50 μL) were in turn injected into a preconditioned Acquity BEH C18 (1.7 μm, 2.1 × 50 m) silica-based, reverse phase column, on a Waters Acquity UPLC system (Waters, Mississauga, ON). The flow rate was 0.25 mL/min, and a linear increase from zero to 28% ACN in water with 0.1% formic acid was used. Mass spectrometric detection was performed on a Waters G2 Synapt ESI-MS instrument. Positive ionization mode was used. The analyzer mode was set to high resolution, with a capillary voltage of 3.00 kV and a cone voltage of 25 V. The Progenesis™ software was used to handle and search databases for these HPLC-MS/MS analyses.
Separations were conducted on a LC Ultra system (Eksigent, Dublin, CA). A 100 μm × 200 mm analytical column packed with 3 μm Luna C18 (Phenomenex, Torrance, CA) was used, at 500 nL/min flow rate. A 300 μm × 5 mm PepMap 100 trap-column (Thermo Fisher, San Jose, CA) was used to protect the analytical column. The elution gradient was as described above. A Triple TOF 5600 mass spectrometer (ABSciex, Concord, ON) was used in standard MS/MS data-dependent acquisition mode. Mass spectra (250 ms) were collected (
In previous studies, wild-type porcine IgG was digested with trypsin, and glycopeptides/peptides were fractionated on a C18 cartridge. Glycopeptide fractions were analyzed by MALDI-MS/MS [2, 3, 30]. In a new separate experiment, the results of which are presented here, all tryptic products were then enriched for glycopeptides using an EMD-Millipore ProteoGlycan™ kit [23]. The glycopeptide-enriched fraction was injected into a RPLC-MS/MS system, using data-dependent MS/MS acquisition. Figure 1a shows the MS/MS total ion chromatogram (TIC) obtained from the elution, whereas Figure 1b represents the elution of peptides with
(a) Total HPLC/MS ion chromatogram obtained from the MS/MS spectra of porcine IgG tryptic peptides and glycopeptides after enrichment on EMD-Millipore Proteoglycan™. (b) Selected MS/MS fragment ion chromatogram at
A database search using GPM [24] helped to identify some non-glycosylated peptides, while for glycopeptides the extracted
Peptide sequence | Error (ppm) | Glycoform | Identification source* | ||
---|---|---|---|---|---|
Unknown | 646.35 | Unavailable | |||
NFSTYR | 0 | 2231.14 2392.28 2701.08 2863.10 | G0F G1F G1FS G2FS | IgG1a-b,IgG2a-b,IgG4a-b,IgG5a-b, IgG6a-b,L8B0S7,L8B0S2,K7ZLA7,L8B180, L8B0Z4 | |
LLVELIR | 855.57 | 4.44 | Unavailable | ||
TVTPSECA | 864.38 | 3.8 | P01846 | ||
FSGAISGNK | 880.45 | 2.72 | P01846 | ||
DLPAPTIR | 882.5 | −4.88 | IgG2ba-b,IgG4a-b,IgG6a-b,L8B180,L8B0S7,L8B0Z4 | ||
LLLDLFR | 889.55 | −0.56 | Unavailable | ||
LLNGYRR | 891.52 | −4.6 | Unavailable | ||
AGGTTVTQVE | 962.48 | 1.14 | P01846 | ||
LIYQATNR | 978.54 | 3.37 | IPR 007110 (Ig C1-set) | ||
VDPALPLEK | 981.56 | −1.58 | Unavailable | ||
NRPTGVPSR | 983.54 | −1.9 | Unavailable | ||
TISKATGPSR | 1017.57 | 1.28 | IgG3 | ||
LSSPATLNSR | 1045.56 | −3.44 | Unavailable | ||
Unknown | 2933.2 | M9 | Unavailable | ||
FQQTPGQPPP | 1096.54 | −2.09 | P01846 | ||
EAQFNSTYR | −19.4 | 2414.05 2560.12 2722.15 2884.24 3029.35 3191.50 | G0 G0F G1F G2F G1FS G3F | IgG6a,L8B180,L8B0Z4 | |
Unknown | 2599.1 | G0F | Unavailable | ||
EEQFNSTYR | −14.6 | 2471.01 2618.09 2634.07 2780.13 3087.23 2942.19 3104.23 3249.28 2833.04 | G0 G0F G1 G1F G1FS G2F G3F G2FS M9-N | IgG1a-b,IgG2a-b,IgG4a-b,IgG5ab,IgG6b, L8B0S7,L8B0S2,K7ZLA7 | |
TNNRPTGVPSR | 1198.63 | Unavailable | |||
H2N-300-QNFSVFR | 2656.09 2819.14 2981.20 | G0F G1F G2F | Unavailable | ||
SYLALSASDWK | 1240.62 | −0.64 | P01846 | ||
DTNRPSGIPER | 1241.62 | −2.6 | Unavailable | ||
STNSRPTGVPSR | 1258.65 | 0.16 | P01846 | ||
FSGSGSGTDFTLK | 1303.62 | 2.74 | Unavailable | ||
SSSGFTCQVTHE | 1339.56 | 1.27 | P01846 | ||
TAPSVYPLAPCGR | 1388.71 | 7.85 | IgG1a-b,IgG4b,IgG5b,IgG6b,L8B180,K7ZLA7,L8B0Z4 | ||
LLGASVLGVGDIHR | 1406.81 | 1 | Unavailable | ||
Unknown | 1415.62 | Unavailable | |||
Unknown | 2911.26 3073.31 | G0F G1F | Unavailable | ||
Unknown | 1495.73 | Unavailable | |||
LVESGGGLVQPGGSLR | 1525.85 | 11 | L8B0S2,L8B180,L8B0S7 | ||
QSNNKYAASSYLAL | 1529.76 | 0.39 | P01846 | ||
AGGTTVTQVETTKPSK | 1604.85 | 0.6 | P01846 | ||
YAASSYLALSASDWK | 1632.79 | 0.24 | P01846 | ||
VVSVLPIQHQDWLK | 1661.92 | −10.4 | IgG1a-b,IgG3,IgG6b,L8B180,K7ZLA7,L8B0Z4 | ||
QEYREDFVLTVTGK | 1667.83 | 12.7 | Unavailable | ||
APASYFVQSVLTVSAK | 1667.9 | 0 | K7ZJP7 | ||
Unknown | 1718.91 | Unavailable | |||
TVIYSTNSRPTGVPSR | 1734.91 | 1.73 | P01846 | ||
QLIYSTNNRPTGVPSR | 1802.95 | 3.88 | Unavailable | ||
QLIYQTNSRPTGVPSR | 1816.97 | 1.98 | IPR 007110 (Ig-like) | ||
SSSGFTCQVTHEGTIVEK | 1966.92 | 1.32 | P01846 | ||
AAPTVNLFPPSSEELGTNK | 1972 | 1.06 | P01846 | ||
ASGVPDRFSGSGSGTDFTLK | 1985.96 | 1.86 | Unavailable | ||
FTDETLVSDLQPSLDRAR | 2063.04 | 0.15 | Unavailable | ||
ATLVCLISDFYPGAVTVWK | 2083.1 | 3.26 | P01846 | ||
AGPLGWFERRPEPPPGPPSK | 2172.14 | 2.39 | Unavailable | ||
QSNNKYAASSYLALSASDWK | 2204.06 | 0.82 | P01846 | ||
Unknown | 2717.07 | Unavailable | |||
Unknown | 2832.04 | Unavailable |
Masses and sequences of peptides found in a glycopeptide-enriched fraction from porcine IgG (wild-type) tryptic digestion products. Red: Labeled manually; black: Sequenced and/or matched by GPM [24]. Glycopeptides,
Points of interest arising from this table are (i) the presence of a large number of non-glycosylated, even after enrichment, from the Fab and Fc portions of the antibody and (ii) the detection of seven distinct glycosylated peptides, some with complex glycoforms and one with high-mannose glycoforms, thus of the
Figure 2 shows four MS/MS spectra of glycopeptides, starting with two of the complex G0F forms of peptides of (a) constant region EAQFNSTYR [3] and (b) a variable region sequence partially determined as H2N-(300)-QNFSVFR by the CycloBranch software [26]. In these cases, the mass difference between the protonated precursors and (M + H)+ bare peptide fragments is 1444. The
Tandem mass spectra of doubly or triply charged ions of glycopeptides enriched from the tryptic products of porcine wild-type IgG. (a) G0F glycoform of EAQFNSTYR, (b) G0F of undetermined peptide, (c) G1FS of EEQFNSTYR, (d) Man-9 of undetermined peptide.
For known peptide sequences such as in Figure 2a and c, it is possible to find most bare peptide y and b ions, although they appear with very low abundance and are not accounted for by the search engine, due to the domination of all glycopeptide signature ions. There was an attempt by the authors to sequence all unknown glycopeptide sequences, with partial success, as indicated in red in Table 1.
Overall results suggest that
Antibody samples were first fragmented on immobilized papain, and thus it is expected that only IgG-related products will be present in the mixture. As shown by Figure 3a, all antibody was fragmented (intact antibody would have appeared at ca. 4.5 min). In general, when IgG is incubated with papain with a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragments and one Fc fragment. The Fc may remain intact based on conditions and enzyme used [9, 10]. The cleavages occur at cysteines around position 271 (in Figure A1), about 10 amino acids from the IdeS cleavage site [29]. In human IgG subtypes (IgG1-4), there are on average of three cysteines in the range of hinge region positions 265–275 to make the numbers correspond with those of Figure A1, where papain cleavages can be initiated [29]. Porcine IgG has similar cysteine motifs in these positions; however, Figure A1 shows different lengths of amino acid chains in the hinge region, which did not seem to prevent fragmentation. Interestingly in this papain-fragmentation experiment, the Fc did not remain intact, but the Fab did. Figure 3a shows significant separation although not at the baseline, but which still allowed the collection of Fab and Fc fractions.
Size exclusion chromatograms obtained for 200 μg of wild-type porcine IgG fragmented by (a) immobilized papain and (b) Fabulous™.
In order to further isolate Fab and Fc segments, collected fractions were analyzed in turn by SDS-PAGE. This allowed identification by mass, with confirmation by linear mode MALDI-TOF-MS (not shown) and in-gel tryptic digestion of the bands (analyzed by reflector mode MALDI-TOF-MS and HPLC-MS, next section). The first major peak in Figure 3a was identified as originating from the Fab, and the second peak was identified as the Fc. The intact Fab fragment is larger and thus elutes prior to the split Fc, while the opposite is usually true in the case of human IgG [15].
Fabulous™ is a recombinant cysteine protease which under reducing conditions digests in the hinge region of antibodies from many species and subclasses, including human, mouse, rat, and rhesus monkey, yielding Fab and Fc fragments [11]. Some specific fragmentation site information is available for human IgG1, mouse IgG1, and rabbit IgG, but no information at all pertains to the fragmentation of porcine IgG. Looking at all porcine IgG subtype sequences (see alignment in Figure A1), they have a motif similar to that of rabbit IgG (KP270I/CPP) [11], that is, with a potential fragmentation site between isoleucine I and cysteine C: CP270I/CPG or CP270I/CPA.
In general, when IgG molecules are incubated with Fabulous™ in the presence of a reducing agent, one or more peptide bonds in the hinge region are split next to a cysteine, producing two Fab fragments and one Fc. As the reducing agent is present during the digestion reaction, it is likely that all interchain thiols will be reduced.
In this experiment with Fabulous™ using 100 μg of pig IgG, a small portion of the antibody was not fragmented; otherwise the Fc was split into two halves and the Fab fragments remained intact. The HPLC-SEC chromatogram is shown in Figure 3b. The first major peak was indicative of the Fab fragment, and the second peak eluting after identified as the Fc, as verified later by SDS-PAGE and further tryptic digestion. As noticed in the chromatograms of Figure 3, elution times are different, that is, longer in portion b. This is due to gradual deterioration of the column, as both experiments depicted in a and b were performed several months apart.
The fact that Fabulous™ (28,724 Da [11]) was free in solution and not immobilized as in the case of papain involves that it would elute in the SEC chromatogram (Figure 3b), most probably in the second peak with the Fc. If this proprietary enzyme has a sequence similar to that of papain, subsequent proteolysis by trypsin is likely to occur extensively, as many lysine and arginine residues are present in papain’s sequence [31].
For papain-produced fragments, a nonreducing gel experiment was performed on the previously collected SEC fractions (Figure 4a). This experiment allowed confirming the identity of the fragments. A reducing SDS-PAGE experiment was also performed on intact pig IgG to serve as a control (Figure 4b). Bands were then excised from the gel, followed by in-gel tryptic digestion. This helped to single out heavy and light chains for differentiating between Fab and Fc according to their respective known glycopeptides and peptides [2, 3]. In Figure 4a, lanes 6–9 correspond to a fraction containing both Fab and Fc collected at the junction of SEC peaks in Figure 3a. A single 50 kDa Fab band and two 25 kDa bands were obtained. The Fab band is positioned just below the 50 kDa marker band and clearly below the Fc band in Figure 4b, and the Fab should have a lower molecular weight than intact Fc [32]. Both bands appearing in the Fc region were digested independently with trypsin as denoted by “upper Fc band” and “lower Fc band.” It appears from further results that they belong to different IgG subtypes. For Fabulous™ fragments, the same experiment was conducted and results are shown in Figure 4b. According to gel separations, papain and Fabulous™ had very similar fragmentation effects on porcine IgG.
(a) Fab and Fc SEC fractions from papain-fragmented porcine IgG on nonreducing SDS-PAGE and (b) from Fabulous™-fragmented porcine IgG, (c) whole porcine IgG runs under reducing conditions.
Figure 5a shows the tryptic products for the upper Fc band (papain generated) in Figure 4a. The main glycopeptide observed has three main glycoforms at
Reflector positive mode MALDI-TOF-MS spectra of (a) tryptic digestion products from pig IgG Fc fragment (higher 25 kDa band) and (b) lower Fc fragment. Both bands were excised from the gel run under non-reducing conditions shown in
Peptide sequence | Error ppm | Glyco-form | Source* | ||
---|---|---|---|---|---|
Unknown | 807.28 | Unavailable | |||
Unknown | 842.38 | Unavailable | |||
Unknown | 870.41 | Unavailable | |||
DLPAPITR | 882.5 | −5.78 | IgG2ba-b,IgG4a-b,IgG6a-b, L8B180,L8B0S7,L8B0Z4 | ||
Unknown | 905.33 | Unavailable | |||
Unknown | 951.33 | Unavailable | |||
Unknown | 993.36 | Unavailable | |||
Unknown | 1033.4 | Unavailable | |||
Unknown | 1107.4 | Unavailable | |||
EAQFNSTYR | −22.51 | 2560.39 | G0F | IgG6a,L8B180,L8B0Z4 | |
Unknown | 1165.4 | Unavailable | |||
EEQFNSTYR | −14.6 | 2471.60 2779.62 2779.63 | G0 G0F G1F | IgG1a-b,IgG2a-b,IgG4a-b,IgG5a-b, IgG6b,L8B0S7,L8B0S2,K7ZLA7 | |
Unknown | 1209.5 | Unavailable | |||
Unknown | 1261.4 | Unavailable | |||
VNNVDLPAPITR | 1308.6 | −66.51 | IgG1a-b,K7ZLA7 | ||
Unknown | 1330.4 | Unavailable | |||
SNGQPEPEGNYR | 1347.6 | −1.48 | IgG1a,IgG6a-b,L8B180,L8B0S2, K7ZLA7,L8B0Z4 | ||
Unknown | 1374.4 | Unavailable | |||
Unknown | 1392.2 | Unavailable | |||
Unknown | 1427.6 | Unavailable | |||
Unknown | 1475.6 | Unavailable | |||
Unknown | 1503.4 | Unavailable | |||
LVESGGGLVQPGGSLR | 1525.7 | −74.23 | L8B180,L8B0S7 | ||
VSSQNIQDFPSVLR | 1589.8 | 42.9 | K7ZJP7 (IgM HC const. region) | ||
YAASSYLALSASDWK | 1632.8 | −24.5 | P01846 UniprotKB (Ig λ const. region) | ||
VVSVLPIQHQDWLK | 1662 | 13.7 | IgG1a-b,IgG3,IgG6b,L8B180,K7ZLA7, L8B0Z4 | ||
Unknown | 1677.5 | Unavailable | |||
Unknown | 1693.7 | Unavailable | |||
Unknown | 1725.4 | Unavailable | |||
Unknown | 1740.6 | Unavailable | |||
Unknown | 1754.4 | Unavailable | |||
STGKPTLYNVSLVLSDT | 1794.8 | −60.4 | K7ZJP7 (IgM HC const. region) | ||
EPQVYTLSPSAEELSR | 1805.8 | −34.1 | IgG6a-b,L8B180,L8B0Z4 | ||
EPQVYTLPPPAEELSR | 1825.9 | 2.9 | IgG1a-b,K7ZLA7 | ||
EPQVYTLPPPTEELSR | 1855.9 | −2.32 | IgG4b | ||
TTPPQQDVDGTFFLYSK | 1943.8 | −50.7 | IgG1a-b,K7ZLA7 | ||
TTPPQQDVDGTYFLYSK | 1959.9 | −1.07 | IgG2a-b,IgG3,IgG4a-b,IgG6a,L8B180, L8B0S7,L8B0Z4 | ||
AAPTVNLFPPSSEELGTNK | 1971.9 | −36.6 | P01846 UniprotKB (Ig λ const. region) | ||
Unknown | 2135.8 | Unavailable | |||
Unknown | 2162.9 | Unavailable | |||
Unknown | 2211 | Unavailable | |||
GLEGLAYIGYTGVITDYADSVK | 2305.4 | 86.8 | L8B180 |
Masses and sequences of peptides found by MALDI-MS in porcine IgG (wild-type) tryptic digestion products of combined Fc bands obtained by papain and Fabulous™ fragmentation. Peptides and glycopeptides in red were sequenced manually after MS/MS and/or tentatively identified by mass fingerprinting.
The bands just below 50 kDa from both papain and Fabulous™ digestion processes were excised from the gel and digested with trypsin. MALDI-MS spectra of the products were obtained, and display no obvious glycopeptides as found in the Fc. Some peaks could be identified as light and heavy chain Fab peptides. Fab peptide masses and sequences available are listed in Table 3.
Sequence | Error ppm | Identification source | ||
---|---|---|---|---|
FSGAISGNK | 880.45 | 880.39 | −70.8 | P01846 (Ig λ const. region) |
Unknown | 951.33 | 951.21 | Unavailable | |
Unknown | 993.36 | 993.56 | Unavailable | |
Unknown | 1052.22 | Unavailable | ||
Unknown | 1119.29 | Unavailable | ||
Unknown | 1141.26 | Unavailable | ||
Unknown | 1209.48 | 1209.28 | Unavailable | |
Unknown | 1242.51 | Unavailable | ||
FSGSGSGTDFTLK | 1303.62 | 1303.61 | −4.91 | Unavailable |
Unknown | 1339.55 | Unavailable | ||
Unknown | 1374.27 | Unavailable | ||
Unknown | 1383.95 | Unavailable | ||
Unknown | 1419.30 | Unavailable | ||
Unknown | 1476.02 | Unavailable | ||
Unknown | 1503.39 | 1504.12 | Unavailable | |
Unknown | 1526.39 | Unavailable | ||
Unknown | 1584.35 | Unavailable | ||
Unknown | 1622.45 | Unavailable | ||
YAASSYLALSASDWK | 1632.79 | 1632.85 | 36.7 | P01846 (Ig λ const. region) |
Unknown | 1660.16 | Unavailable | ||
Unknown | 1735.19 | Unavailable | ||
Unknown | 1762.23 | Unavailable | ||
QLIYSTNNRPTGVPSR | 1802.95 | 1803.00 | 27.73 | Unavailable |
Unknown | 1826.41 | Unavailable | ||
Unknown | 1866.38 | Unavailable | ||
Unknown | 1910.24 | Unavailable | ||
Unknown | 1942.26 | Unavailable | ||
AAPTVNLFPPSSEELGTNK | 1972.00 | 1972.18 | 90.2 | P01846 (Ig λ const. region) |
Unknown | 2034.37 | Unavailable | ||
FTDETLVSDLQPSLDRAR | 2063.04 | 2063.17 | 62.9 | Unavailable |
Unknown | 2135.81 | 2136.40 | Unavailable | |
Unknown | 2210.95 | 2211.46 | Unavailable | |
Unknown | 2338.64 | Unavailable | ||
Unknown | 2377.63 | Unavailable | ||
Unknown | 2408.38 | Unavailable | ||
VTLTCLVTGFYPPDIDVEWQR | 2509.24 | 2509.39 | 58.5 | IgG4a |
Unknown | 2691.50 | Unavailable | ||
Unknown | 2807.49 | Unavailable |
Overall, MALDI-TOF-MS experiments allowed suggesting that efficient separation of Fab and Fc fragments from each other occurred, using the SEC/SDS-PAGE procedure. There was a minimal number of overlapping peptides between Fab and Fc MALDI spectra (Tables 2 and 3). In Table 3, peptides in red had already been sequenced and appeared in Table 1; peptides whose
Each sample of digestion products from Fc and Fab bands were analyzed twice by HPLC/MS, and results are summarized in Table 4. In Figure A2, total ion chromatograms (TIC) of the pig Fab and Fc tryptic products showed differences in the retention times of some peaks as expected. Peaks observed at the same retention times (e.g., 4.41–4.46, 10.35, and 23.10 min) and common to all injections corresponded to singly charged ions of small compounds of
Mass (calc.) | Normalized abundance | Sequence | Sources of identification* | |||
---|---|---|---|---|---|---|
516.301 | 517.309 | 0.00 | 0.0 | 23.9 | VDKR | IgG1a-b,IgG2a-b,IgG3,IgG4a-b |
585.322 | 586.330 | −1.97 | 76.1 | 1972.6 | PGGSLR | P01786 |
712.372 | 713.380 | −4.25 | 8.0 | 891.4 | LVESGGGL | P01786 |
768.412 | 769.420 | 0.0 | 9.4 | |||
806.345 | 807.353 | 652.0 | 22.4 | |||
811.439 | 812.447 | −5.63 | 6.1 | 424.4 | LVESGGGLV | P01786 |
816.416 | 817.424 | 0.0 | 78.4 | |||
824.372 | 825.380 | 0.0 | 9.1 | |||
826.434 | 827.442 | 749.4 | 1176.6 | |||
827.511 | 828.519 | 478.3 | 166.0 | |||
834.421 | 835.429 | 0.0 | 65.1 | |||
837.492 | 838.500 | −5.12 | 14.2 | 0.0 | ALPAPIEK | P01857 |
841.502 | 842.510 | 977.9 | 1260.9 | |||
844.291 | 845.299 | 264.0 | 0.0 | |||
850.418 | 851.426 | 0.0 | 42.4 | |||
861.432 | 862.440 | 220.3 | 52.3 | |||
872.373 | 873.381 | 0.0 | 74.7 | |||
872.397 | 873.405 | 0.0 | 15.5 | |||
879.445 | 880.453 | −0.45 | 1559.7 | 479.8 | FSGAISGNK | P01846 |
881.492 | 882.500 | −5.78 | 0.0 | 410.6 | DLPAPITR | IgG2ba-b,IgG4a-b, IgG6a-b,L8B180,L8B0S7,L8B0Z4 |
888.366 | 889.374 | 0.0 | 44.9 | |||
917.391 | 918.399 | 254.9 | 54.6 | |||
919.450 | 920.458 | 17.9 | 135.1 | |||
935.856 | 936.864 | 2502.6 | 2690.0 | |||
939.510 | 940.518 | 7.61 | 86.4 | 407.8 | LVESGGGLVQ | P01786 |
990.341 | 991.349 | 264.4 | 5.6 | |||
994.506 | 995.513 | 13.2 | 518.7 | |||
994.512 | 995.520 | 7.6 | 311.7 | |||
1043.899 | 1044.907 | 1589.5 | 1690.0 | |||
1044.554 | 1045.562 | −2.30 | 5106.9 | 6511.7 | LSSPATLNSR | Unavailable |
1057.831 | 1058.839 | 1179.7 | 1190.5 | |||
1066.539 | 1067.547 | −1.47 | 0.0 | 8.5 | VDGVEVHNAK | P01857 |
1103.601 | 1104.609 | 0.0 | 17.0 | |||
1164.577 | 1165.585 | 66.4 | 199.4 | |||
1174.613 | 1175.621 | −5.01 | 468.4 | 169.5 | TQGVETTKPSK | P01846 |
1208.640 | 1209.648 | 0.0 | 790.8 | |||
1238.648 | 1239.656 | 1.6 | 343.5 | |||
1259.553 | 1260.561 | 3.7 | 480.5 | |||
1260.536 | 1261.544 | 15.5 | 1386.4 | |||
1273.685 | 1274.693 | −1.89 | 324.0 | 104.5 | VTQGVETTKPSK | P01846 |
1296.678 | 1297.686 | 9.3 | 727.0 | |||
1298.485 | 1299.493 | 30.8 | 263.2 | |||
1302.609 | 1303.617 | 0.00 | 2446.2 | 908.9 | FSGSGSGTDFTLK | Unavailable |
1307.718 | 1308.726 | −1.30 | 139.6 | 13772.8 | VNNVDLPAPITR | IgG1a-b,K7ZLA7 |
1308.702 | 1309.710 | 13.2 | 3997.2 | |||
1312.672 | 1313.680 | −1.22 | 2.4 | 306.7 | ESGGGLVQPGGSLR | P01786 |
1316.625 | 1317.633 | 896.9 | 223.7 | |||
1329.687 | 1330.695 | 20.3 | 434.9 | |||
1330.670 | 1331.678 | 87.9 | 1324.0 | |||
1334.648 | 1335.656 | 23.1 | 374.1 | |||
1340.548 | 1341.556 | 131.8 | 45.0 | |||
1345.666 | 1346.674 | 5.8 | 2149.3 | |||
1346.587 | 1347.595 | 1.41 | 47.2 | 658.2 | SNGQPEPEGNYR | IgG1a,IgG5b,IgG6b,L8B180K7ZLA7,L8B0Z4 |
1347.568 | 1348.576 | 50.8 | 1702.0 | |||
1350.620 | 1351.628 | 7.9 | 512.8 | |||
1368.616 | 1369.624 | 16.4 | 484.9 | |||
1374.732 | 1375.740 | −2.30 | 742.1 | 219.7 | TVTQGVETTKPSK | P01846 |
1376.558 | 1377.566 | 457.8 | 61.0 | |||
1390.608 | 1391.616 | 3.4 | 308.0 | |||
1391.593 | 1392.601 | 6.6 | 898.0 | |||
1399.808 | 1400.816 | 1434.9 | 1503.7 | |||
1433.682 | 1434.690 | 8.5 | 226.8 | |||
1449.688 | 1450.696 | 17.8 | 958.3 | |||
1514.667 | 1515.675 | 100.8 | 1436.0 | |||
1524.822 | 1525.830 | −2.62 | 1005.8 | 20216.6 | LVESGGGLVQPGGSLR | L8B180, L8B0S7 |
1530.664 | 1531.672 | 0.3 | 599.0 | |||
1532.803 | 1533.811 | −0.91 | 646.1 | 171.1 | GTTVTQGVETTKPSK | P01846 |
1546.798 | 1547.806 | 64.1 | 2405.4 | |||
1562.772 | 1563.780 | 192.8 | 4750.7 | |||
1578.744 | 1579.752 | 47.70 | 4.7 | 439.3 | EEQFNSTYR +2GlcNAc | IgG1a-b,IgG2a-b,IgG4a-b,IgG5a-b,IgG6b,L8B0S7,L8B0S2,K7ZLA7 |
1589.823 | 1590.831 | −1.93 | 647.8 | 181.7 | GGTTVTQGVETTKPSK | P01846 |
1624.841 | 1625.849 | 321.8 | 72.8 | |||
1642.851 | 1643.859 | −0.92 | 1827.2 | 614.7 | AGGTTVTQGVETTKPSK | [C-term] neutral loss |
1660.864 | 1661.872 | 0.26 | 11688.6 | 4028.5 | AGGTTVTQGVETTKPSK | P01846 |
1682.829 | 1683.837 | 346.8 | 77.9 | |||
1698.811 | 1699.819 | 1146.9 | 308.5 | |||
1719.703 | 1720.711 | 10.5 | 471.9 | |||
1735.702 | 1736.710 | 0.0 | 480.4 | |||
1753.805 | 1754.813 | 2.7 | 493.2 | |||
1769.797 | 1770.805 | 4.8 | 723.1 | |||
1801.950 | 1802.958 | 3.60 | 191.0 | 99.2 | QLIYSTNNRPTGVPSR | Unavailable |
1807.752 | 1808.760 | 4.8 | 287.3 | |||
1824.931 | 1825.939 | 2.90 | 4.0 | 670.0 | EPQVYTLPPPAEELSR | IgG1a-b,K7ZLA7 |
1854.932 | 1855.940 | −2.32 | 0.0 | 1499.6 | EPQVYTLPPPTEELSR | IgG4b |
1862.867 | 1863.875 | 0.0 | 695.8 | |||
1919.930 | 1920.938 | 1.8 | 0.0 | |||
1958.924 | 1959.932 | −1.07 | 3.4 | 2082.2 | TTPPQQDVDGTYFLYSK | IgG4a-b, IgG6a, L8B180, L8B0S7, IgG2a, IgG2b, IgG3 |
2092.889 | 2093.897 | 649.7 | 14.8 | |||
2251.915 | 2252.923 | 0.0 | 750.1 | |||
2267.910 | 2268.918 | 0.0 | 356.8 | |||
2289.908 | 2290.916 | 0.0 | 72.8 | |||
2355.931 | 2356.939 | 0.0 | 62.0 | |||
2413.960 | 2414.968 | −11.42 | 0.0 | 2793.9 | G0 of EAQFNSTYR | IgG6a,L8B180,L8B0Z4 |
2435.939 | 2436.947 | 0.0 | 86.5 | |||
2451.911 | 2452.919 | 0.0 | 90.0 | |||
2558.988 | 2559.995 | −19.69 | 0.0 | 25.6 | G0F EAQFNSTYR | IgG6a,L8B180,L8B0Z4 |
2576.008 | 2577.016 | 0.0 | 439.0 | |||
2617.036 | 2618.044 | −2.66 | 3.5 | 1257.9 | G0F EEQFNSTYR | IgG1a-b,IgG2a-b,IgG4a-b,IgG5a-b,IgG6b,L8B0S7,L8B0S2,K7ZLA7 |
2639.006 | 2640.014 | 0.0 | 706.3 | |||
2654.984 | 2655.992 | 2.5 | 1048.1 | |||
2670.955 | 2671.963 | 0.0 | 200.5 | |||
2692.958 | 2693.966 | 0.0 | 49.2 | |||
2779.092 | 2780.100 | −1.36 | 0.0 | 1327.0 | G1F EEQFNSTYR | IgG1a-b,IgG2a-b,IgG4a-b,IgG5a-b,IgG6b,L8B0S7,L8B0S2,K7ZLA7 |
2801.070 | 2802.078 | 2.8 | 244.1 | |||
2817.033 | 2818.041 | 0.0 | 380.8 | |||
2941.166 | 2942.174 | 5.90 | 0.0 | 266.4 | G2F EEQFNSTYR | IgG1a-b,IgG2a-b,IgG4a-b,IgG5a-b,IgG6b,L8B0S7,L8B0S2,K7ZLA7 |
3004.216 | 3005.224 | 0.0 | 68.7 | |||
3086.182 | 3087.190 | −1.33 | 0.0 | 159.4 | G1FS EEQFNSTYR | IgG1a-b,IgG2a-b,IgG4a-b,IgG5a-b,IgG6b,L8B0S7,L8B0S2,K7ZLA7 |
Tryptic peptides from pig IgG Fab and Fc fragment analyzed by HPLC-MS/MS.
As Fabulous™ was possibly still present in the mixture at the time of tryptic digestion of the Fc gel band, analogous papain tryptic digestion products were sought for by
The initial RPLC-MS/MS experiment on the enriched whole IgG digest produced the most useful information in terms of glycopeptides. Besides the latter, many peptides were identified as originating from the Fc or Fab portion, while for other peptides origin remained unknown. This first workflow allowed detecting
The authors thank Dr. Jean-Paul Soulillou and his team at the Université de Nantes, France, for providing pig IgG samples. Thanks to Genovis for donating Fabulous™ enzyme.
None of the authors has a conflict of interest.
Alignment of sequences available in the literature for the porcine IgG heavy chains. IgGn: from Ref. [
HPLC/MS total ion chromatograms obtained for the tryptic products of wild-type porcine IgG Fabulous™ fragments, (a) Fc and (b) Fab.
Porcine immunoglobulins constitute a complex ensemble of biomolecules, with several subtypes whose amino acid sequences are not clearly assigned and described in the literature (see Figure A1). In this study, three state-of-the-art mass spectrometers were used to characterize tryptic peptides, offering a considerable amount of complementary information owing to the great sensitivities of these instruments, given the small amounts of IgGs used. Indeed, fragmentation of porcine IgG into its Fc and Fab portions was achieved for the first time using papain and Fabulous™ on 200 μg or less of antibody. Fragments needed separation by both SEC and SDS-PAGE before analysis by MALDI-TOF-MS and HPLC/MS. These separation procedures did not eliminate the overlapping of Fab and Fc tryptic peptides entirely; however, there was a significant level of discrimination. This workflow resulted in better knowledge about the origin of tryptic peptides from IgG.
During the course of this work, the several sources of sequence information on porcine IgG found in Figure A1 were used to verify HPLC-MS/MS, MALDI-TOF-MS, and HPLC-MS data. Most Fc peptides were from the gamma constant region, where some Fab peptides were identified as belonging to the constant portion of the lambda chain. The HPLC-MS/MS method of tryptic peptide without previous Fab-Fc fragmentation was the most efficient in terms of useful data generated per amount of sample used, although many peptides could not be related to either Fc or Fab. This study highlights the need for detailed pertinent sequence information for porcine IgG, which is not a commonly studied set of biomolecules. Future work will involve the quantification of IgG subtypes according to unique peptide sequences that are already known in each subtype.
Nanotechnology involves synthesizing and developing different nanomaterials. The field of nanotechnology allows different nanoparticles of unique features to be produced. Nanoparticles (NPs) are complex material particles that fall within the range of one to hundred nanometers. Their nanometer sizes drive the chemical, optical, physical, and electric features of the nanoparticles [1]. Naturally, nanoparticles can be sourced from geological, biological, meteorological, and cosmological means. However, nanoparticles can be created from liquid and solid materials by breaking down biopolymers, condensing gases, wet chemical process, implantation of ions, hydrothermal process, pyrolysis, radiolysis etc. Nanoparticles are usually viewed with the aid of electron microscopes, can penetrate filters, and have unique mechanical properties that distinguish them from the bulk materials. Nanoparticles exist in various shapes like nanorods, nanostars, nanofibers, nanospheres, nanoflowers, nanoboxes etc. [2].
Nanoparticles comprise a functionalized surface, a shell of different layered materials, and the core/main nanoparticle [3]. The features of materials in their bulk form are different from their nanoparticle forms because of the large area to volume ratio, interfacial layer, affinity to solvents, kind of coating, quantum mechanics effects, rate of diffusion, mechanical, and ferromagnetic features [1]. The large area to volume ratio makes the nanoparticles highly reactive and able to penetrate membranes. The chemical nature of nanoparticles should be studied to enhance their molecular attachment to surfaces.
Nanoparticles may be metallic, non-metallic [1], anthropogenic, engineered, organic, or inorganic as outlined in Figure 1. Metallic nanoparticles include copper, magnesium, zinc, gold, titanium, silver etc.; while non-metallic nanoparticles include silica, carbon nanotubes etc. Anthropogenic NPs are by-products obtained from industrial produce while engineered nanoparticles are directly obtained from manufacturing processes.
Schematics on the classifications of nanoparticles.
Some of the nanoparticles and their features [2, 4] have been summarized in Table 1.
Nanoparticles | Features |
---|---|
Silver | Very effective, high antimicrobial performance, wide range of usage |
Gold | Good for identifying protein interactions, useful in tracing out fingerprints, detects antibiotics and cancerous cells, efficient for cancer diagnosis and other bacteria |
Iron | Biocompatible and useful for treating cancer, sorting stem cells, analyzing genes, and drug delivery |
Quantum dot | Diameters less than 10 nm, semiconducting nanoparticle, size-dependent |
Carbon nanotubes | sp2 hybridized carbon atoms, strong electron bonds, high electrical conductivity, good catalysts. |
Copper | Wide absorption spectrum, distinct optical features, yields good quality nanoparticles |
Ceramics | Inorganic amorphous solids; could be polycrystalline, porous, amorphous or dense; vastly applied in photocatalysis, imaging devices etc. |
Semiconductor | Large and tunable band gap nature, suitable in water splitting and electronic appliances. |
Polymeric | Majorly organic components and easily functionalized |
Lipid-based | Comprise lipid components, uses surfactants as core stabilizers |
Some nanoparticles and their respective features.
The techniques applied in synthesizing nanoparticles greatly influence their morphology, size, structure, and performance. The electrochemical, physiochemical, optical, and electrical features of the nanoparticles are also affected. In some occasions, nanoparticles are coated so as to retain their features after precipitating out of suspensions. The synthesis methods for nanoparticles are broadly divided into top-down and bottom-up approaches [4].
Top-down method is a destructive method that breaks down large molecules into smaller parts before converting into the relevant nanoparticles. This approach involves some decomposition strategies like chemical vapor deposition (CVD), milling process, and physical vapor deposition (PVD). Milling is used to extract nanoparticles from coconut shells with the crystallite size reducing with increasing time. Nanoparticles of iron oxide, carbon, dichalcogenides, cobalt (III) oxide have been produced using this method.
This approach involves the formation of nanoparticles from simple materials in a build-up manner. It is environmentally friendly, less poisonous, feasible, and of low cost. The materials used are usually Reduction and sedimentation processes like green synthesis, bio-chemical, spin coating, sol–gel etc. adopt this approach. Nanoparticles of titanium dioxide, gold, bismuth have been synthesized via this approach. The reaction chain for the production of gold nanoparticle has been illustrated in Figure 2 [5].
Formation process of gold nanoparticle.
Synthesizing nanoparticles could also involve chemical or biological processes [1]. Some chemical synthesis techniques of nanoparticles include sol–gel method, wet chemical synthesis, hydrothermal method, thermal decomposition, microwave method etc. [2]; while the biological means involve enzymes, microorganisms, plant extracts, and fungi.
Some chemical methods adopted in synthesizing nanoparticles include sol gel, precipitation, hydrothermal, thermal decomposition, solvothermal, vapor synthesis etc. [6, 7]. Sol–gel method is an easy means of producing nanostructures by homogenously mixing precursors in a solvent to form a gel material which is then heated to produce the required nanoparticle. It begins from preparing a sol which undergoes gelation process to solvent removal. Wet chemical/precipitation method is a fast and easy process for synthesizing large scale nanoparticles. Hydrothermal method utilizes high pressure and temperature to power heterogeneous reactions under aqueous solvents like water. The kind of pressure, pH, and temperature applied affects the features of the synthesized nanoparticles. Such nanoparticles are suitable for biotechnological use because of their hydrophilic surface nature [8]. Thermal decomposition involves oxidizing a solid material in optimal temperature. Solvothermal method uses a solvent to produce various materials like polymers, semiconductors, or metals at moderate or high pressure [9]. It produces novel and stable nanoparticles with controlled thicknesses and temperature. To synthesize nanodots; the cationic source is dissolved in suitable solvent alongside a surfactant which stabilizes the growth rate. Cadmium selenide, zinc oxide, zinc selenide are producible using this method and can be applied in magnetic and biotech industries [10]. In vapor synthesis, gaseous molecules chemically react to produce a phase which condenses and leads to particle growth. The higher the temperature, the faster the particles are formed. Different means of inducing homogenous nucleation include condensing inert gases, vaporizing a supersaturated material using a pulsed laser, generating a spark discharge by charging electrodes, sputtering the material with unreactive gaseous ions; or through some chemical methods like chemical vapor deposition, photothermal method, flame synthesis, or spray pyrolysis [11]. This method suitably yields nanoparticles of titania, carbon, and silica. Flame synthesis is commonly used to commercially produce silica, carbon black, optical fiber, and titania [12]. Particles produced by converting gases in furnace reactors or hot walls are usually very pure, although it produces agglomerated particles.
Biological or biosynthesis of nanoparticles is an environmentally-friendly, green, and non-toxic method involving microorganisms [13, 14, 15]. Nanoparticles of iron oxide, silver, nickel oxide, copper oxide, zinc ferrite have been synthesized using this method [16, 17, 18, 19, 20, 21, 22]. The location of the nanoparticle determines the point of synthesis; whether intracellular or extracellular [1]. Intracellular production of nanoparticles uses enzymes to move ions into the cells of microbes and produces smaller sized nanoparticles in the organism. Extracellular synthesis does not involve cell components and yields nanoparticles outside the cell, uses fungi with large secretory organs. Microbes like fungi and bacteria are responsible for controlling the synthesis process. Microorganisms are immensely used to produce nanoparticles because of their economical, non-poisonous nature, and detoxification of heavy metal power. Phytonanotechnology is compatible with biological systems, available source materials, high stability, and entails synthesizing nanoparticles from plants [23]. Changes in the pH level of plants alter their binding strength, morphology, and the number of metallic ions available during the synthesis. The different sources, synthesis methods, and areas of application of nanoparticles have been represented in Figure 3 [23]. Biogenic means of producing nanoparticles are green and cheap; with the involvement of fungi, waste materials, and bacteria [5].
Diverse bio-development synthesis of nanoparticles and their application areas.
Nanoparticles can also be synthesized by mechanical methods like mechanical alloying, milling, and mechanochemical processes [24]. Milling method regenerates interfacial chemical operations at low temperatures. Mechanochemical technique involves continuous welding operations that adequately select milling materials and minimize agglomerations. For effective production; the stoichiometry of source materials, thermal treatment, paths for reaction to occur, and milling conditions would be carefully considered. Nanoparticles of oxides, iron, nickel, silver, cobalt can be synthesized using these methods.
Properties of nanoparticles like shape, size, surface morphology, crystalline nature, light absorption etc. need to be completely described using relevant characterization techniques [2]. Some of the methods used to characterize nanoparticles [4] include:
The morphology of nanoparticles greatly influence the properties exhibited by nanoparticles. Microscopy methods applied on nanoparticles are usually electron microscopy or scanning probe microscopy. Scanning electron microscope (SEM) gives nanoscale and surface information of the dispersion and morphology of nanoparticles. Microscopy techniques are destructive and used for single-particle measurements. Transmission electron microscopy (TEM) uses transmittance of electrons to provide bulk information at high and low magnifications. Optical microscopic technique is not useful for nanoparticles because the size of nanoparticles is smaller than light diffraction limit. Coupling spectroscopic techniques to electron microscopes would enable elemental studies to be carried out.
Optical methods reveal reflectance, transmittance, photochemical, and luminescence features of nanoparticles. Spectroscopy uses the interaction of particles with electromagnetic radiation to determine the shape, concentration, and size of nanoparticles. Spectroscopic techniques like infrared, ultraviolet–visible, photoluminescence (PL), UV/vis-diffuse reflectance spectrometer (DRS), and magnetic resonance methods are applied to nanoparticles. DRS is specially used to determine the band gap energy of nanoparticles. PL studies reveal the effect of emissivity and absorptivity on the excitation of photons, half-life, and recombining effects of the charges. The sizes of nanoparticles affect their optical features and make it useful in bioimaging devices [4].
The structure of nanoparticles gives details about the kind of bond existing between the atoms and the features of the bulk material. Some of the structural techniques used on nanoparticles include BET, X-ray diffractometry (XRD), IR etc. XRD describes the phase, particle size, type of NP, and crystal nature of the nanoparticles.
The elemental composition of nanoparticles can be determined using energy dispersive X-ray spectroscopy (EDX), XPS, Raman, FT-IR etc. EDX details the elemental components of bulk particles. Better contrast is obtainable when the obtained spectra are compared with a computer generated model. XPS is a very sensitive spectroscopic method used to obtain the exact compositional ratio of the elements, their bonding nature, depth profile analysis. Raman and FTIR techniques use vibrational methods to show functionalized peaks and particle information.
Sizes of nanoparticles can be estimated using scanning electron microscope, transmission electron microscope, X-ray diffractometer, atomic force microscope etc. The sizes of the nanoparticles are obtained using size distribution profiles and give more precise results when used alongside digital models. The surface area can be estimated using BET via adsorption and desorption processes.
Mechanical properties, optical activity, surface area, and chemical reactions of nanoparticles are physiochemical characteristics obtainable from nanoparticles. Free surface electrons on nanoparticles are very mobile and are not scattered upon light illumination. The magnetic features of NPs are manifested at small nanoscales due to their uneven distribution, influenced by the synthesis technique adopted, and find vast application in biomedicine, resonance imaging, and catalytic devices. Mechanical characteristics of nanoparticles like stress, surface coatings, hardness, strain, friction, adhesiveness etc. aid an understanding of NPs and greatly affect the quality of the surface. Nanoparticles have great conduction to heat especially on the surface.
Generally, nanoparticles have been applied in various areas including anticancer drugs, vaccines, disease treatment, cancer diagnosis, mechanical factories, electronics, optical devices, energy harvesters, manufacturing processes, cell imaging, and delivery systems due to their unique features [4]. NPs also aid water contaminants to be absorbed on the surface during water purification, serve as environmental sensors, and protect materials from harmful substances. Some of the application areas of the nanoparticles [2, 23] have been summarized in Table 2.
Nanoparticles | Application areas |
---|---|
Nickel oxide | Dye sensitized solar cells, supercapacitors, batteries, water treatment and catalytic systems, gas sensing devices. |
Carbon nanotubes | Integrated circuits, electronic components, textile, construction, cosmetics, medicine |
Cerium oxide | Biomedical equipments, electronic appliances, energy devices |
Titanium dioxide | Coatings, water purifiers, paints |
Silver | Clothing, textile industries, food packaging companies, agriculture, automotives, electronics, medicine, and fitness centers. |
Iron | Optical devices, water purifier |
Calcium | Agriculture, automotives, food |
Zinc oxide | Agriculture, automotives, cosmetics, home appliances, food |
Gold | Cosmetics, environmental products, food, medicine |
Palladium | Automotive, electronic appliances, food |
Application areas of some nanoparticles.
Despite the numerous applications of NPs; they suffer from poisonous and harmful body effects which inhaled, ingested, or transferred to the ground and surrounding environs. Nanoparticles are also affected by organic materials which lead to agglomeration. The poisonous effects associated with NP synthesis can be curtailed by adopting green synthesis methods especially in the synthesis of silver, iron, copper, gold nanoparticles amongst others [25]. The synthesis process for silver nanoparticles is as shown in Figure 4 [25].
Experimental diagram for the bioproduction of silver nanoparticles.
Green synthesis involves different capping substances like biomolecules and polysaccharides. Green methods are non-poisonous, environmentally friendly, involve toxic-free solvents, compatible in biological systems, and utilize reagents like sugars, polymers, vitamins, plant extracts [26]. Plant-based extracts like latex, leaf, seed, root, or stem are more suited for bioprocesses as they are cheap, non-complex, easily reproduced, and highly stable. Other sources of waste materials useful for nanoparticle production have been outlined in Figure 5. Models can be developed to minimize the difficulties associated with distributing the size of the particles and NPs synthesis by computing the rates at which the particles get nucleated [11].
Schematic showing different sources of waste material.
The need for environmentally-friendly and stable nanomaterial that would be compatible with biological systems have prompted researchers into the production of nanoparticles. This chapter gives general knowledge on nanoparticles, their classification, merits and demerits, several synthesis and characterization techniques. Nanoparticles have economical and simple manufacturing processes that are classified into top-down method, bottom-up approach, chemical synthesis, biological method, and mechanical process. Several characterization methods of nanoparticles are geared towards understanding the morphological, structural, optical, size, mechanical, and physiochemical features. Each property is obtainable from different machines and using different techniques. The synthesis and characterization methods employed greatly influence the obtained features of the nanoparticles. Nanoparticles find useful application in medicine, drug delivery, cosmetics, optical devices, electronics, solar cell devices etc.
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His studies in robotics lead him not only to a PhD degree but also inspired him to co-found and build the International Journal of Advanced Robotic Systems - world's first Open Access journal in the field of robotics.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"441",title:"Ph.D.",name:"Jaekyu",middleName:null,surname:"Park",slug:"jaekyu-park",fullName:"Jaekyu Park",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/441/images/1881_n.jpg",biography:null,institutionString:null,institution:{name:"LG Corporation (South Korea)",country:{name:"Korea, South"}}},{id:"465",title:"Dr",name:"Christian",middleName:null,surname:"Martens",slug:"christian-martens",fullName:"Christian Martens",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"479",title:"Dr.",name:"Valentina",middleName:null,surname:"Colla",slug:"valentina-colla",fullName:"Valentina Colla",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/479/images/358_n.jpg",biography:null,institutionString:null,institution:{name:"Sant'Anna School of Advanced Studies",country:{name:"Italy"}}},{id:"494",title:"PhD",name:"Loris",middleName:null,surname:"Nanni",slug:"loris-nanni",fullName:"Loris Nanni",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/494/images/system/494.jpg",biography:"Loris Nanni received his Master Degree cum laude on June-2002 from the University of Bologna, and the April 26th 2006 he received his Ph.D. in Computer Engineering at DEIS, University of Bologna. On September, 29th 2006 he has won a post PhD fellowship from the university of Bologna (from October 2006 to October 2008), at the competitive examination he was ranked first in the industrial engineering area. He extensively served as referee for several international journals. He is author/coauthor of more than 100 research papers. He has been involved in some projects supported by MURST and European Community. His research interests include pattern recognition, bioinformatics, and biometric systems (fingerprint classification and recognition, signature verification, face recognition).",institutionString:null,institution:null},{id:"496",title:"Dr.",name:"Carlos",middleName:null,surname:"Leon",slug:"carlos-leon",fullName:"Carlos Leon",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Seville",country:{name:"Spain"}}},{id:"512",title:"Dr.",name:"Dayang",middleName:null,surname:"Jawawi",slug:"dayang-jawawi",fullName:"Dayang Jawawi",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Technology Malaysia",country:{name:"Malaysia"}}},{id:"528",title:"Dr.",name:"Kresimir",middleName:null,surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/528/images/system/528.jpg",biography:"K. Delac received his B.Sc.E.E. degree in 2003 and is currentlypursuing a Ph.D. degree at the University of Zagreb, Faculty of Electrical Engineering andComputing. His current research interests are digital image analysis, pattern recognition andbiometrics.",institutionString:null,institution:{name:"University of Zagreb",country:{name:"Croatia"}}},{id:"557",title:"Dr.",name:"Andon",middleName:"Venelinov",surname:"Topalov",slug:"andon-topalov",fullName:"Andon Topalov",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/557/images/1927_n.jpg",biography:"Dr. Andon V. Topalov received the MSc degree in Control Engineering from the Faculty of Information Systems, Technologies, and Automation at Moscow State University of Civil Engineering (MGGU) in 1979. He then received his PhD degree in Control Engineering from the Department of Automation and Remote Control at Moscow State Mining University (MGSU), Moscow, in 1984. From 1985 to 1986, he was a Research Fellow in the Research Institute for Electronic Equipment, ZZU AD, Plovdiv, Bulgaria. In 1986, he joined the Department of Control Systems, Technical University of Sofia at the Plovdiv campus, where he is presently a Full Professor. He has held long-term visiting Professor/Scholar positions at various institutions in South Korea, Turkey, Mexico, Greece, Belgium, UK, and Germany. And he has coauthored one book and authored or coauthored more than 80 research papers in conference proceedings and journals. 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Aalborg University has Two Satellite Campuses, one in Copenhagen (Aalborg University Copenhagen) and the other in Esbjerg (Aalborg University Esbjerg).\n· He is a member of prestigious IEEE (Institute of Electrical and Electronics Engineers), and IAENG (International Association of Engineers) organizations. \n· He is the chief Editor of the Journal of Software Engineering.\n· He is the member of the Editorial Board of International Journal of Computer Science and Software Technology (IJCSST) and International Journal of Computer Engineering and Information Technology. \n· He is also the Editor of Communication in Computer and Information Science CCIS-20 by Springer.\n· Reviewer For Many Conferences\nHe is the lead person in making collaboration agreements between Aalborg University and many universities of Pakistan, for which the MOU’s (Memorandum of Understanding) have been signed.\nProfessor Akbar is working in Academia since 1990, he started his career as a Lab demonstrator/TA at the University of Sussex. After finishing his P. hD degree in 1992, he served in the Industry as a Scientific Officer and continued his academic career as a visiting scholar for a number of educational institutions. In 1996 he joined National University of Science & Technology Pakistan (NUST) as an Associate Professor; NUST is one of the top few universities in Pakistan. In 1999 he joined an International Company Lineo Inc, Canada as Manager Compiler Group, where he headed the group for developing Compiler Tool Chain and Porting of Operating Systems for the BLACKfin processor. The processor development was a joint venture by Intel and Analog Devices. In 2002 Lineo Inc., was taken over by another company, so he joined Aalborg University Denmark as an Assistant Professor.\nProfessor Akbar has truly a multi-disciplined career and he continued his legacy and making progress in many areas of his interests both in teaching and research. 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Fungal infectious illness prevalence and prognosis are determined by the exposure between fungi and host, host immunological state, fungal virulence, and early and accurate diagnosis and treatment. \r\nPatients with both congenital and acquired immunodeficiency are more likely to be infected with opportunistic mycosis. Fungal infectious disease outbreaks are common during the post- disaster rebuilding era, which is characterised by high population density, migration, and poor health and medical conditions.\r\nSystemic or local fungal infection is mainly associated with the fungi directly inhaled or inoculated in the environment during the disaster. The most common fungal infection pathways are human to human (anthropophilic), animal to human (zoophilic), and environment to human (soilophile). Diseases are common as a result of widespread exposure to pathogenic fungus dispersed into the environment. \r\nFungi that are both common and emerging are intertwined. In Southeast Asia, for example, Talaromyces marneffei is an important pathogenic thermally dimorphic fungus that causes systemic mycosis. Widespread fungal infections with complicated and variable clinical manifestations, such as Candida auris infection resistant to several antifungal medicines, Covid-19 associated with Trichoderma, and terbinafine resistant dermatophytosis in India, are among the most serious disorders. \r\nInappropriate local or systemic use of glucocorticoids, as well as their immunosuppressive effects, may lead to changes in fungal infection spectrum and clinical characteristics. Hematogenous candidiasis is a worrisome issue that affects people all over the world, particularly ICU patients. CARD9 deficiency and fungal infection have been major issues in recent years. Invasive aspergillosis is associated with a significant death rate. Special attention should be given to endemic fungal infections, identification of important clinical fungal infections advanced in yeasts, filamentous fungal infections, skin mycobiome and fungal genomes, and immunity to fungal infections.\r\nIn addition, endemic fungal diseases or uncommon fungal infections caused by Mucor irregularis, dermatophytosis, Malassezia, cryptococcosis, chromoblastomycosis, coccidiosis, blastomycosis, histoplasmosis, sporotrichosis, and other fungi, should be monitored. \r\nThis topic includes the research progress on the etiology and pathogenesis of fungal infections, new methods of isolation and identification, rapid detection, drug sensitivity testing, new antifungal drugs, schemes and case series reports. It will provide significant opportunities and support for scientists, clinical doctors, mycologists, antifungal drug researchers, public health practitioners, and epidemiologists from all over the world to share new research, ideas and solutions to promote the development and progress of medical mycology.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/4.jpg",keywords:"Emerging Fungal Pathogens, Invasive Infections, Epidemiology, Cell Membrane, Fungal Virulence, Diagnosis, Treatment"},{id:"5",title:"Parasitic Infectious Diseases",scope:"Parasitic diseases have evolved alongside their human hosts. In many cases, these diseases have adapted so well that they have developed efficient resilience methods in the human host and can live in the host for years. Others, particularly some blood parasites, can cause very acute diseases and are responsible for millions of deaths yearly. Many parasitic diseases are classified as neglected tropical diseases because they have received minimal funding over recent years and, in many cases, are under-reported despite the critical role they play in morbidity and mortality among human and animal hosts. The current topic, Parasitic Infectious Diseases, in the Infectious Diseases Series aims to publish studies on the systematics, epidemiology, molecular biology, genomics, pathogenesis, genetics, and clinical significance of parasitic diseases from blood borne to intestinal parasites as well as zoonotic parasites. We hope to cover all aspects of parasitic diseases to provide current and relevant research data on these very important diseases. In the current atmosphere of the Coronavirus pandemic, communities around the world, particularly those in different underdeveloped areas, are faced with the growing challenges of the high burden of parasitic diseases. At the same time, they are faced with the Covid-19 pandemic leading to what some authors have called potential syndemics that might worsen the outcome of such infections. Therefore, it is important to conduct studies that examine parasitic infections in the context of the coronavirus pandemic for the benefit of all communities to help foster more informed decisions for the betterment of human and animal health.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/5.jpg",keywords:"Blood Borne Parasites, Intestinal Parasites, Protozoa, Helminths, Arthropods, Water Born Parasites, Epidemiology, Molecular Biology, Systematics, Genomics, Proteomics, Ecology"},{id:"6",title:"Viral Infectious Diseases",scope:"The Viral Infectious Diseases Book Series aims to provide a comprehensive overview of recent research trends and discoveries in various viral infectious diseases emerging around the globe. The emergence of any viral disease is hard to anticipate, which often contributes to death. A viral disease can be defined as an infectious disease that has recently appeared within a population or exists in nature with the rapid expansion of incident or geographic range. This series will focus on various crucial factors related to emerging viral infectious diseases, including epidemiology, pathogenesis, host immune response, clinical manifestations, diagnosis, treatment, and clinical recommendations for managing viral infectious diseases, highlighting the recent issues with future directions for effective therapeutic strategies.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/6.jpg",keywords:"Novel Viruses, Virus Transmission, Virus Evolution, Molecular Virology, Control and Prevention, Virus-host Interaction"}],annualVolumeBook:{},thematicCollection:[],selectedSeries:null,selectedSubseries:null},seriesLanding:{item:{id:"11",title:"Biochemistry",doi:"10.5772/intechopen.72877",issn:"2632-0983",scope:"Biochemistry, the study of chemical transformations occurring within living organisms, impacts all areas of life sciences, from molecular crystallography and genetics to ecology, medicine, and population biology. Biochemistry examines macromolecules - proteins, nucleic acids, carbohydrates, and lipids – and their building blocks, structures, functions, and interactions. Much of biochemistry is devoted to enzymes, proteins that catalyze chemical reactions, enzyme structures, mechanisms of action and their roles within cells. Biochemistry also studies small signaling molecules, coenzymes, inhibitors, vitamins, and hormones, which play roles in life processes. Biochemical experimentation, besides coopting classical chemistry methods, e.g., chromatography, adopted new techniques, e.g., X-ray diffraction, electron microscopy, NMR, radioisotopes, and developed sophisticated microbial genetic tools, e.g., auxotroph mutants and their revertants, fermentation, etc. More recently, biochemistry embraced the ‘big data’ omics systems. Initial biochemical studies have been exclusively analytic: dissecting, purifying, and examining individual components of a biological system; in the apt words of Efraim Racker (1913 –1991), “Don’t waste clean thinking on dirty enzymes.” Today, however, biochemistry is becoming more agglomerative and comprehensive, setting out to integrate and describe entirely particular biological systems. The ‘big data’ metabolomics can define the complement of small molecules, e.g., in a soil or biofilm sample; proteomics can distinguish all the comprising proteins, e.g., serum; metagenomics can identify all the genes in a complex environment, e.g., the bovine rumen. This Biochemistry Series will address the current research on biomolecules and the emerging trends with great promise.",coverUrl:"https://cdn.intechopen.com/series/covers/11.jpg",latestPublicationDate:"May 18th, 2022",hasOnlineFirst:!0,numberOfOpenTopics:4,numberOfPublishedChapters:287,numberOfPublishedBooks:27,editor:{id:"31610",title:"Dr.",name:"Miroslav",middleName:null,surname:"Blumenberg",fullName:"Miroslav Blumenberg",profilePictureURL:"https://mts.intechopen.com/storage/users/31610/images/system/31610.jpg",biography:"Miroslav Blumenberg, Ph.D., was born in Subotica and received his BSc in Belgrade, Yugoslavia. He completed his Ph.D. at MIT in Organic Chemistry; he followed up his Ph.D. with two postdoctoral study periods at Stanford University. Since 1983, he has been a faculty member of the RO Perelman Department of Dermatology, NYU School of Medicine, where he is codirector of a training grant in cutaneous biology. Dr. Blumenberg’s research is focused on the epidermis, expression of keratin genes, transcription profiling, keratinocyte differentiation, inflammatory diseases and cancers, and most recently the effects of the microbiome on the skin. He has published more than 100 peer-reviewed research articles and graduated numerous Ph.D. and postdoctoral students.",institutionString:null,institution:{name:"New York University Langone Medical Center",institutionURL:null,country:{name:"United States of America"}}},subseries:[{id:"14",title:"Cell and Molecular Biology",keywords:"Omics (Transcriptomics; Proteomics; Metabolomics), Molecular Biology, Cell Biology, Signal Transduction and Regulation, Cell Growth and Differentiation, Apoptosis, Necroptosis, Ferroptosis, Autophagy, Cell Cycle, Macromolecules and Complexes, Gene Expression",scope:"The Cell and Molecular Biology topic within the IntechOpen Biochemistry Series aims to rapidly publish contributions on all aspects of cell and molecular biology, including aspects related to biochemical and genetic research (not only in humans but all living beings). We encourage the submission of manuscripts that provide novel and mechanistic insights that report significant advances in the fields. Topics include, but are not limited to: Advanced techniques of cellular and molecular biology (Molecular methodologies, imaging techniques, and bioinformatics); Biological activities at the molecular level; Biological processes of cell functions, cell division, senescence, maintenance, and cell death; Biomolecules interactions; Cancer; Cell biology; Chemical biology; Computational biology; Cytochemistry; Developmental biology; Disease mechanisms and therapeutics; DNA, and RNA metabolism; Gene functions, genetics, and genomics; Genetics; Immunology; Medical microbiology; Molecular biology; Molecular genetics; Molecular processes of cell and organelle dynamics; Neuroscience; Protein biosynthesis, degradation, and functions; Regulation of molecular interactions in a cell; Signalling networks and system biology; Structural biology; Virology and microbiology.",annualVolume:11410,isOpenForSubmission:!0,coverUrl:"https://cdn.intechopen.com/series_topics/covers/14.jpg",editor:{id:"165627",title:"Dr.",name:"Rosa María",middleName:null,surname:"Martínez-Espinosa",fullName:"Rosa María Martínez-Espinosa",profilePictureURL:"https://mts.intechopen.com/storage/users/165627/images/system/165627.jpeg",institutionString:null,institution:{name:"University of Alicante",institutionURL:null,country:{name:"Spain"}}},editorTwo:null,editorThree:null,editorialBoard:[{id:"79367",title:"Dr.",name:"Ana Isabel",middleName:null,surname:"Flores",fullName:"Ana Isabel Flores",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRpIOQA0/Profile_Picture_1632418099564",institutionString:null,institution:{name:"Hospital Universitario 12 De Octubre",institutionURL:null,country:{name:"Spain"}}},{id:"328234",title:"Ph.D.",name:"Christian",middleName:null,surname:"Palavecino",fullName:"Christian Palavecino",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y000030DhEhQAK/Profile_Picture_1628835318625",institutionString:null,institution:{name:"Central University of Chile",institutionURL:null,country:{name:"Chile"}}},{id:"186585",title:"Dr.",name:"Francisco Javier",middleName:null,surname:"Martin-Romero",fullName:"Francisco Javier Martin-Romero",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bSB3HQAW/Profile_Picture_1631258137641",institutionString:null,institution:{name:"University of Extremadura",institutionURL:null,country:{name:"Spain"}}}]},{id:"15",title:"Chemical Biology",keywords:"Phenolic Compounds, Essential Oils, Modification of Biomolecules, Glycobiology, Combinatorial Chemistry, Therapeutic peptides, Enzyme Inhibitors",scope:"Chemical biology spans the fields of chemistry and biology involving the application of biological and chemical molecules and techniques. In recent years, the application of chemistry to biological molecules has gained significant interest in medicinal and pharmacological studies. This topic will be devoted to understanding the interplay between biomolecules and chemical compounds, their structure and function, and their potential applications in related fields. Being a part of the biochemistry discipline, the ideas and concepts that have emerged from Chemical Biology have affected other related areas. This topic will closely deal with all emerging trends in this discipline.",annualVolume:11411,isOpenForSubmission:!0,coverUrl:"https://cdn.intechopen.com/series_topics/covers/15.jpg",editor:{id:"441442",title:"Dr.",name:"Şükrü",middleName:null,surname:"Beydemir",fullName:"Şükrü Beydemir",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y00003GsUoIQAV/Profile_Picture_1634557147521",institutionString:null,institution:{name:"Anadolu University",institutionURL:null,country:{name:"Turkey"}}},editorTwo:{id:"13652",title:"Prof.",name:"Deniz",middleName:null,surname:"Ekinci",fullName:"Deniz Ekinci",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002aYLT1QAO/Profile_Picture_1634557223079",institutionString:null,institution:{name:"Ondokuz Mayıs University",institutionURL:null,country:{name:"Turkey"}}},editorThree:null,editorialBoard:[{id:"241413",title:"Dr.",name:"Azhar",middleName:null,surname:"Rasul",fullName:"Azhar Rasul",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRT1oQAG/Profile_Picture_1635251978933",institutionString:null,institution:{name:"Government College University, Faisalabad",institutionURL:null,country:{name:"Pakistan"}}},{id:"178316",title:"Ph.D.",name:"Sergey",middleName:null,surname:"Sedykh",fullName:"Sergey Sedykh",profilePictureURL:"https://mts.intechopen.com/storage/users/178316/images/system/178316.jfif",institutionString:null,institution:{name:"Novosibirsk State University",institutionURL:null,country:{name:"Russia"}}}]},{id:"17",title:"Metabolism",keywords:"Biomolecules Metabolism, Energy Metabolism, Metabolic Pathways, Key Metabolic Enzymes, Metabolic Adaptation",scope:"Metabolism is frequently defined in biochemistry textbooks as the overall process that allows living systems to acquire and use the free energy they need for their vital functions or the chemical processes that occur within a living organism to maintain life. Behind these definitions are hidden all the aspects of normal and pathological functioning of all processes that the topic ‘Metabolism’ will cover within the Biochemistry Series. Thus all studies on metabolism will be considered for publication.",annualVolume:11413,isOpenForSubmission:!0,coverUrl:"https://cdn.intechopen.com/series_topics/covers/17.jpg",editor:{id:"138626",title:"Dr.",name:"Yannis",middleName:null,surname:"Karamanos",fullName:"Yannis Karamanos",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002g6Jv2QAE/Profile_Picture_1629356660984",institutionString:null,institution:{name:"Artois University",institutionURL:null,country:{name:"France"}}},editorTwo:null,editorThree:null,editorialBoard:[{id:"243049",title:"Dr.",name:"Anca",middleName:null,surname:"Pantea Stoian",fullName:"Anca Pantea Stoian",profilePictureURL:"https://mts.intechopen.com/storage/users/243049/images/system/243049.jpg",institutionString:null,institution:{name:"Carol Davila University of Medicine and Pharmacy",institutionURL:null,country:{name:"Romania"}}},{id:"203824",title:"Dr.",name:"Attilio",middleName:null,surname:"Rigotti",fullName:"Attilio Rigotti",profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institutionString:null,institution:{name:"Pontifical Catholic University of Chile",institutionURL:null,country:{name:"Chile"}}},{id:"300470",title:"Dr.",name:"Yanfei (Jacob)",middleName:null,surname:"Qi",fullName:"Yanfei (Jacob) Qi",profilePictureURL:"https://mts.intechopen.com/storage/users/300470/images/system/300470.jpg",institutionString:null,institution:{name:"Centenary Institute of Cancer Medicine and Cell Biology",institutionURL:null,country:{name:"Australia"}}}]},{id:"18",title:"Proteomics",keywords:"Mono- and Two-Dimensional Gel Electrophoresis (1-and 2-DE), Liquid Chromatography (LC), Mass Spectrometry/Tandem Mass Spectrometry (MS; MS/MS), Proteins",scope:"With the recognition that the human genome cannot provide answers to the etiology of a disorder, changes in the proteins expressed by a genome became a focus in research. Thus proteomics, an area of research that detects all protein forms expressed in an organism, including splice isoforms and post-translational modifications, is more suitable than genomics for a comprehensive understanding of the biochemical processes that govern life. The most common proteomics applications are currently in the clinical field for the identification, in a variety of biological matrices, of biomarkers for diagnosis and therapeutic intervention of disorders. From the comparison of proteomic profiles of control and disease or different physiological states, which may emerge, changes in protein expression can provide new insights into the roles played by some proteins in human pathologies. Understanding how proteins function and interact with each other is another goal of proteomics that makes this approach even more intriguing. Specialized technology and expertise are required to assess the proteome of any biological sample. Currently, proteomics relies mainly on mass spectrometry (MS) combined with electrophoretic (1 or 2-DE-MS) and/or chromatographic techniques (LC-MS/MS). MS is an excellent tool that has gained popularity in proteomics because of its ability to gather a complex body of information such as cataloging protein expression, identifying protein modification sites, and defining protein interactions. The Proteomics topic aims to attract contributions on all aspects of MS-based proteomics that, by pushing the boundaries of MS capabilities, may address biological problems that have not been resolved yet.",annualVolume:11414,isOpenForSubmission:!0,coverUrl:"https://cdn.intechopen.com/series_topics/covers/18.jpg",editor:{id:"200689",title:"Prof.",name:"Paolo",middleName:null,surname:"Iadarola",fullName:"Paolo Iadarola",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bSCl8QAG/Profile_Picture_1623568118342",institutionString:null,institution:{name:"University of Pavia",institutionURL:null,country:{name:"Italy"}}},editorTwo:{id:"201414",title:"Dr.",name:"Simona",middleName:null,surname:"Viglio",fullName:"Simona Viglio",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRKDHQA4/Profile_Picture_1630402531487",institutionString:null,institution:{name:"University of Pavia",institutionURL:null,country:{name:"Italy"}}},editorThree:null,editorialBoard:[{id:"72288",title:"Dr.",name:"Arli Aditya",middleName:null,surname:"Parikesit",fullName:"Arli Aditya Parikesit",profilePictureURL:"https://mts.intechopen.com/storage/users/72288/images/system/72288.jpg",institutionString:null,institution:{name:"Indonesia International Institute for Life Sciences",institutionURL:null,country:{name:"Indonesia"}}},{id:"40928",title:"Dr.",name:"Cesar",middleName:null,surname:"Lopez-Camarillo",fullName:"Cesar Lopez-Camarillo",profilePictureURL:"https://mts.intechopen.com/storage/users/40928/images/3884_n.png",institutionString:null,institution:{name:"Universidad Autónoma de la Ciudad de México",institutionURL:null,country:{name:"Mexico"}}},{id:"81926",title:"Dr.",name:"Shymaa",middleName:null,surname:"Enany",fullName:"Shymaa Enany",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRqB9QAK/Profile_Picture_1626163237970",institutionString:null,institution:{name:"Suez Canal University",institutionURL:null,country:{name:"Egypt"}}}]}]}},libraryRecommendation:{success:null,errors:{},institutions:[]},route:{name:"profile.detail",path:"/profiles/435321",hash:"",query:{},params:{id:"435321"},fullPath:"/profiles/435321",meta:{},from:{name:null,path:"/",hash:"",query:{},params:{},fullPath:"/",meta:{}}}},function(){var e;(e=document.currentScript||document.scripts[document.scripts.length-1]).parentNode.removeChild(e)}()