3,657 research outputs found

    Single muscle fiber proteomics reveals unexpected mitochondrial specialization

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    Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity

    De novo sequencing of proteins by mass spectrometry

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    Introduction Proteins are crucial for every cellular activity and unraveling their sequence and structure is a crucial step to fully understand their biology. Early methods of protein sequencing were mainly based on the use of enzymatic or chemical degradation of peptide chains. With the completion of the human genome project and with the expansion of the information available for each protein, various databases containing this sequence information were formed. Areas covered De novo protein sequencing, shotgun proteomics and other mass-spectrometric techniques, along with the various software are currently available for proteogenomic analysis. Emphasis is placed on the methods for de novo sequencing, together with potential and shortcomings using databases for interpretation of protein sequence data. Expert opinion As mass-spectrometry sequencing performance is improving with better software and hardware optimizations, combined with user-friendly interfaces, de-novo protein sequencing becomes imperative in shotgun proteomic studies. Issues regarding unknown or mutated peptide sequences, as well as, unexpected post-translational modifications (PTMs) and their identification through false discovery rate searches using the target/decoy strategy need to be addressed. Ideally, it should become integrated in standard proteomic workflows as an add-on to conventional database search engines, which then would be able to provide improved identification.publishe

    Proteomics for rejection diagnosis in renal transplant patients: where are we now?

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    Rejection is one of the key factors that determine the long-term allograft function and survival in renal transplant patients. Reliable and timely diagnosis is important to treat rejection as early as possible. Allograft biopsies are not suitable for continuous monitoring of rejection. Thus, there is an unmet need for non-invasive methods to diagnose acute and chronic rejection. Proteomics in urine and blood samples has been explored for this purpose in 29 studies conducted since 2003. This review describes the different proteomic approaches and summarizes the results from the studies that examined proteomics for the rejection diagnoses. The potential limitations and open questions in establishing proteomic markers for rejection are discussed, including ongoing trials and future challenges to this topic

    Proteomic Tools for Food and Feed Authentication

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    På grunn av globalt økende etterspørsel etter mat og fôr, introduseres nye proteinholdige ingredienser i matsystemene våre i økende skala. Innføring av nye ingredienser og introduksjon av sirkulære matsystemer gir nye utfordringer når det gjelder metoder for avsløring av henholdsvis fôr- og matsvindel. I denne sammenhengen er det viktig å utvikle raske, sensitive og robuste molekylære metoder som kan implementeres i kontroll og overvåkningsøyemed. Tidligere har fremskritt ved bruk av slike verktøy blitt hemmet av en generell mangel på annoterte referansegenomer for målarter som ofte brukes, eller nylig er introdusert, i fôr eller matpreparater. Fokuset for denne doktorgraden er å utvikle og implementere massespektrometriske metoder (LC-MS/MS) som er i stand til å identifisere, differensiere og kvantifisere proteinholdige ingredienser av animalsk og planteopprinnelse i ulike mat- og fôrblandinger ved bruk av massespektra fingeravtrykk. Arbeidet som presenteres i denne doktorgraden omfatter «bottom-up» proteomiske arbeidsflyter ved bruk av høytrykksvæskekromatografi (HPLC) tandem massespektrometri (MS/MS). Databehandling ble utført ved å bruke direkte spektrasammenligning (compareMS2) og spektrabibliotekmatching (SLM) analyser ved bruk av verktøy fra Trans-Proteomics Pipeline (TPP) og annen åpen kildekode til bioinformatisk programvare. Alle data generert og publisert i løpet av denne doktorgraden har blitt gjort tilgjengelig på offentlige repositrium for MS-data, for eksempel Mass Spectrometry Interactive Virtual Environment (MassIVE), som følger FAIR-prinsippene. Den SLM baserte arbeidsflyten brukt i denne doktorgraden klarte å differensiere ulike prosesserte animalske proteiner (PAP) som storfemelk og bovint blod. SLM ble også brukt til å differensiere ulike insektarter og for å detektere om larver av svart soldatflue (BSF) var fôret med PAP. SLM-metoden ble også brukt til å identifisere og kvantifisere innholdet i et blandingsprodukt av 3 ulike fiskearter. Det ble også funnet at SLM basert proteomikk kan brukes til å identifisere vanlige allergener i insektsprøver tiltenkt humant konsum. Denne tilnærmingen ble også implementert med suksess for å differensiere mellom soyabønneprøver som var enten dyrket organisk, konvensjonelt eller inneholdt genetiske modifikasjoner (GM). I tillegg ble differensiell proteinekspresjon påvist mellom prøver av GM, konvensjonelt og økologisk dyrkede soyabønner. Dette førte til identifisering av to nye peptidmarkører for effektiv sporing av GM-avlinger i mat og fôr. Denne doktorgraden har vist at den SLM baserte metoden er i stand til å identifisere både art og vevstype brukt i et proteinholdig matprodukt eller fôringredients det være seg PAP, plante-, pattedyr- eller fiskeproteiner. Fremtidig arbeid bør fokusere på differensiering og avsløring av svindel i sjømat, som nylig ble fremhevet som et fremvoksende tema i det globale matmarkedet. Alle arts- og vevsspesifikke MS-data samlet inn i det ovennevnte arbeidet vil gjøres tilgjengelig fra i dedikert nettbaserte tjenester. Sistnevnte utvikles for tiden internt, og etter skikkelig kvalitetstesting er det tenkt å bli utgitt offentlig for å gi forskningsmiljøer og myndigheter en lett tilgjengelig plattform for autentisering og identifisering av proteinholdige ingredienser i fôr- og mat. Due to globally rising demands for food and feed, novel proteinaceous ingredients are introduced into our food systems on an increasing scale. The introduction of novel ingredients and circularity of the food system gives rise to novel challenges concerning the detection of feed and food fraud and the determination of feed and food authenticity, respectively. In this context, developing and increasing the implementation of rapid, sensitive, and robust molecular methods are essential. In the past, progress in applying such tools has been hampered by a general lack of wellannotated reference genomes of target species commonly used or newly introduced in feed or food preparations. This PhD focused on developing and implementing mass spectrometry-based approaches to identify, differentiate, and quantify proteinaceous ingredients of animal and plant origin in various food and feed mixes without using any genomic information. The work presented in this PhD implemented bottom-up proteomic workflows using high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). Data analyses were done using direct spectra comparison (compareMS2), spectra library matching (SLM), Trans-Proteomics Pipeline (TPP), and MaxQuant software. All data generated and published during this PhD have been made available on public repositories for proteomics data, such as the Mass Spectrometry Interactive Virtual Environment (MassIVE), following Findable, Accessible, Interoperable, and Reusable (FAIR) principles. The untargeted proteomics SLM workflow implemented during this PhD successfully differentiated processed animal proteins such as bovine milk and bovine blood. The SLM was also used to identify and authenticate food and feed-grade insect species and to detect if black soldier fly (BSF) larvae were fed on the prohibited PAP. Using the SLM workflow, it was also possible to quantify and authenticate the different species in fish mixtures containing muscle tissues from three different fish species. It was also shown that untargeted proteomics could be used to identify common allergens in foodgrade insect samples. Also, the proteomic approach was successfully implemented to separate thirty-one ready-to-market soybean samples farmed organically, conventionally, and with genetic modifications (GM). Differential protein expression was detected between GM, conventionally, and organically farmed soybean samples. Additional bioinformatics analyses led to the detection of two novel peptide markers for the efficient tracing of GM crops in food and feed. The proteomic tools implemented during this PhD were capable of species and tissues specific identification of proteinaceous food and feed ingredients, including processed animal proteins, plant, mammalian, and fish proteins. Future work should focus on the differentiation and detection of fraud in food and feed in the global food market. Webbased interphase will be developed for food and feed authentication using spectra libraries created during this PhD. Following proper quality testing, the web-based interphase will be released publicly to provide research and regulatory laboratories with an easily accessible platform for authenticating and identifying protein ingredients in feed and food samples.Doktorgradsavhandlin

    Current challenges in software solutions for mass spectrometry-based quantitative proteomics

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    This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

    Proteomische Analyse des nativen und fibrotischen humanen Lebermatrisoms für Organ Engineering

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    A persistent demand in suitable grafts has underscored the need for the development of bioartificial organs. Despite global effort to innovate novel alternatives, the creation of transplants yet faces fundamental challenges that are related to organ specific functionality and immunogenicity and hinder current approaches from clinical translation. A vital objective to successfully implement bioartificial organs into clinical practice is the creation of complex biomaterials that will mediate regenerative capacities for long-lasting performance of newly developed grafts. In this thesis, I present in-depth analysis of human derived liver extracellular matrices by utilizing a label-free shotgun proteomic approach. By applying various decellularization and defatting strategies to fabricate organotypic tissues for proteomic measurements, this work aims to provide insights into the native human liver matrisome to determine the overall complexity that needs to be taken into account when creating novel biomaterials to be functionalized in the development of bioartificial organs. Furthermore, by utilizing the devised experimental workflow for the analysis of fibrotic and cirrhotic human liver extracellular matrices, proteomic features were detected that can be potential targets for therapeutic exploitation. In addition to these two intertwining research threads, this thesis introduces a versatile platform to further functionalize the created human liver extracellular matrices by enabling a facile combination of these scaffolds with prevailing technology to successfully translate various bioengineering approaches from concept to therapeutic reality. The results obtained from human liver matrices support the necessity of including proteomic techniques in interpreting processes underlying tissue homeostasis and regeneration. Proteomic analysis of native tissues provides cues to be utilized in the creation of bioartificial products. This information shall bridge the gap between currently available digital fabrication technology and the clinical usage of functional organ replacements. Furthermore, by analyzing fibrotic and cirrhotic human derived liver scaffolds, specific characteristics were described that demand further investigation to fully understand signaling pathways underlying the emergence of fibrosis and cirrhosis.Eine anhaltende Nachfrage nach geeigneten Transplantaten hat die Notwendigkeit der Entwicklung bio-artifizieller Organe unterstrichen. Trotz globaler Bemühungen steht die Erzeugung von Transplantaten noch vor grundlegenden Herausforderungen, die mit der organspezifischen Funktionalität und Immunogenität zusammenhängen und aktuelle Ansätze an der klinischen Translation hindern. Ein wichtiges Ziel für die erfolgreiche Einführung bioartifizieller Organe in die klinische Praxis ist die Entwicklung und Herstellung komplexer Biomaterialien, welche zu Regeneration fähig sind und somit eine langanhaltende biologische Leistung ermöglichen. Im Rahmen dieser Dissertation führte ich eine eingehende Analyse von extrazellulären Lebermatrizes humanen Ursprungs unter Verwendung eines label-free shotgun proteomischen Ansatzes durch. Durch die Anwendung verschiedener Dezellularisierungs- und Entfettungsstrategien zur Herstellung organotypischer Gewebe für proteomische Analysen zielt diese Arbeit darauf ab, Einblicke in das native humane Lebermatrisom zu geben, um die gesamte Komplexität zu bestimmen, die bei der Herstellung neuartiger Biomaterialien zur Entwicklung bio-artifizieller Organe zum Einsatz kommen sollte. Des Weiteren wurde der entworfene experimentelle Arbeitsablauf verwendet, um proteomische Merkmale fibrotischer und zirrhotischer humaner Lebermatrizes zu identifizieren, die potenzielle Ziele für eine therapeutische Nutzung sein können. Zusätzlich zu diesen beiden miteinander verknüpften Forschungsfäden wird in dieser Arbeit eine vielseitig anwendbare Plattform zur umfangreichen Funktionalisierung der extrazellulären Lebermatrizes humanen Ursprungs vorgestellt. Hierdurch soll die Kombination dieser Matrizes mit vorherrschenden Technologien erleichtert werden, um die verschiedenen Ansätze des Bioengineerings erfolgreich vom Konzept zur therapeutischen Realität umzusetzen. Die in dieser Arbeit präsentierten Ergebnisse aus humanen Lebermatrizes unterstreichen die Relevanz proteomischer Techniken bei der Interpretation der Prozesse, die der Gewebehomöostase und -regeneration zugrunde liegen. Die Untersuchung der proteomischen Landschaft natürlicher Gewebe liefert Kenntnisse, die in der Erzeugung bioartifizieller Produckte verwendet werden können. Diese Informationen sollen die Lücke zwischen verfügbaren digitalen Fertigungstechnologien und dem klinischen Einsatz funktioneller Organersatzprodukte schließen. Darüber hinaus wurden durch die Analyse von fibrotischen und zirrhotischen humanen Lebermatrizes spezifische Eigenschaften beschrieben, die weitere Untersuchungen erfordern, um die der Entstehung von Fibrose und Zirrhose zugrunde liegenden Signalwege vollständig zu verstehen
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