Name: sampling human indigenous saliva peptidome using a lollipop-like ultrafiltration probe: simplify and enhance peptide detection for clinical mass spectrometry
Uploaded: 2012-08-07T13:00:00
Description: Watch this Scientific Journal Video about Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Diese Arbeit wurde vom National Institutes of Health Grants (R01-AI067395-01, R21-R022754-01 und R21-I58002-01) unterstützt. Wir danken C. Niemeyer für die kritische Durchsicht des Manuskripts.

1. Erstellung von LLUF Probes <ol><li> Die Polyethersulfonmembranen (2 cm 2) wurden mit Polypropylen-Dreieck Paddel (University of California, San Diego) durch Kleben mit Epoxy-Membranen an den Grenzen der Paddel versiegelt. Ein negativ geladenes Polyethersulfonmembran mit einem Molekulargewicht cut-off (MWCO) bei 30 kDa verwendet wurde. </li><li> Ein Teflon fluorierten Ethylen-Propylen-(innerer Durchmesser / äußerer Durchmesser 0.35/0.50 cm) wurde einem Zylinder Ausgang eines Dreiecks Polypropylen Paddel...

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Wir haben festgestellt, dass viele Peptidfragmente in menschlichen unverdaute Speichel abgegeben. Diese Peptidfragmente sind Derivate aus verschiedenen Formen von Prolin-reiche Proteine, Actin, alpha-Amylase, Alpha-1-Globin, beta-Globin, histain 1, Keratin 1, Muzin 7, polymeren Immunglobulin-Rezeptor, satherin, S100A9. Es könnte viele Faktoren, die die Produktion von Peptiden mit ungeklärter Spaltstellen sein. Zum Beispiel können einige Peptidfragmente natürlich vorhanden sein in menschlichem Gesamtblut Speichel. Vi...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Polyethersulfonmembranen </td><td> Pall Corporation </td><td></td><td> 30 kDa MWCO </td></tr><tr><td> Teflon fluorierten Ethylen-Propylen-Rohr </td><td> Upchurch Scientific </td><td></td><td></td></tr><tr><td> Blaudextran </td><td> Sigma </td><td></td><td></td></tr><tr><td> Nano-LC-System </td><td> Eksigent </td><td></td><td></td></tr><tr><td> C18-Trap-Säule </td><td> Agilent </td><td> 5065-9913 </td><td></td></tr><tr><td> LTQ linearen Ionenfallen-Massenspektrometer </td><td> Thermo Fisher </td><td></td><td></td></tr><tr><td> Sorcerer 2 </td><td> Sage-N ReseaRCH </td><td></td><td></td></tr><tr><td> Acetonitril-0.1% Ameisensäure </td><td> JT Baker </td><td> 9832-03 </td><td> LC / MS-Klasse </td></tr><tr><td> Wasser-0,1% Ameisensäure </td><td> JT Baker </td><td> 9834-03 </td><td> LC / MS-Klasse </td></tr></tbody></table>

sampling human indigenous saliva peptidome using a lollipop-like ultrafiltration probe: simplify and enhance peptide detection for clinical mass spectrometry

Obwohl menschlichen Speichel Proteom-und Peptidoms worden 1-2 offenbart haben sie majorly von tryptischen Verdaus von Speichel-Proteine ​​identifiziert. Identifizierung von indigenen Peptidoms von menschlichem Speichel ohne vorherige Spaltung mit exogenen Enzyme wird aber notwendig, da nativen Peptide im menschlichen Speichel mögliche Werte stellen für die Diagnose von Krankheiten, die Vorhersage Fortschreiten der Erkrankung sowie die Überwachung therapeutische Wirksamkeit. Entsprechende Probenahme ist ein wichtiger Schritt für die Verbesserung der Identifikation der menschlichen Speichel indigenen Peptidoms. Traditionelle Methoden der Probenahme menschlichen Speichel mit Zentrifugation, um Verschmutzungen zu entfernen 4.3 kann zu zeitaufwendig anwendbar zu sein für den klinischen Einsatz. Darüber hinaus kann Müllbeseitigung durch Zentrifugation nicht in der Lage, die meisten der infizierten Krankheitserregern reinigen und entfernen Sie die hohe Abundanz Proteine, die oft behindern die Identifikation von geringer Menge Peptidoms. Herkömmliche Ansätze Proteomik, dass priLinie nutzen zweidimensionale Gelelektrophorese (2-DE Gelen) in Konjugation mit in-Gel-Verdau, imstande sind, viele Proteine ​​Speichel 5-6. Allerdings ist dieser Ansatz im Allgemeinen nicht ausreichend empfindlich gegenüber niedrigen Häufigkeit Peptide / Proteine ​​zu detektieren. Flüssigchromatographie-Massenspektrometrie (LC-MS) basierten Proteomik ist eine Alternative, die Proteine ​​ohne vorherige 2-DE Trennung identifizieren können. Obwohl dieser Ansatz bietet eine höhere Empfindlichkeit, muss es in der Regel vor Proben-Fraktionierung 7 und vor dem Verdau mit Trypsin, was es schwierig für den klinischen Gebrauch macht. Um das Hindernis in der Massenspektrometrie durch Probenvorbereitung zu umgehen, haben wir eine Technik, die als Kapillare Ultrafiltration (CUF) Sonden 8-11 entwickelt. Daten aus unserem Labor gezeigt, dass die Sonden, die CUF Erfassung Proteine ​​in vivo aus verschiedenen Mikroumgebungen bei Tieren in einem dynamischen und minimal invasiven Weise 8 -11. Keine Zentrifugation ist notwendig, da ein Unterdruck durch einfaches Herausziehen Spritze während der Probenahme erstellt wird. Die CUF-Sonden mit LC-MS kombiniert erfolgreich identifizierten tryptischen-verdaute Proteine ​​11.08. In dieser Studie wurden aufgerüstet wir die Ultrafiltration Sampling-Technik, indem sie einen Lutscher-wie Ultrafiltration (LLUF) Sonde, die sich leicht in die menschliche Mundhöhle kann passen. Die direkte Analyse mittels LC-MS ohne Trypsin-Verdau zeigte, dass menschliche Speichel enthält viele indigenously Peptidfragmente aus verschiedenen Proteinen abgeleitet sind. Probenahme mit Speichel LLUF Sonden vermieden Zentrifugation aber effektiv entfernt viele größere und hohe Abundanz Proteine. Unsere massenspektrometrischen Ergebnisse dargestellt, dass viele geringer Menge nachweisbar Peptide nach Herausfiltern von größeren Proteinen mit LLUF Sonden wurde. Erkennung von Peptiden geringer Menge Speichel war unabhängig von mehrstufigen Stichprobe Trennung mit Chromatographie. Für die klinische Anwendung, verfügen die Sonden LLUFd mit LC-MS könnte möglicherweise in der Zukunft genutzt werden, um den Krankheitsverlauf aus Speichel zu überwachen.

Watch this Scientific Journal Video about Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry 

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Angesichts Speichel Probenahme für zukünftige klinische Anwendung wurde ein Lollipop-ähnliche Ultrafiltration (LLUF) Sonde hergestellt, um in die menschliche Mundhöhle passen. Direkte Analyse von unverdauten Speichel mit nanoLC-LTQ Massenspektrometrie zeigten die Fähigkeit von LLUF Sonden, um große Proteine ​​und hohe Abundanz Proteine ​​zu entfernen, und machen Low-reiche Peptide mehr nachweisbar.

Probenahme menschliche Speichel Indigene Peptidoms Mit einem Lollipop-Like Ultrafiltration Sonde: vereinfachen und erweitern Peptid-Erkennung für Klinische Massenspektrometrie

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

Research

JoVE Journal

Medicine

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

Mit visuellen und narrativen Methoden auf ein faires Verfahren in der klinischen Versorgung erreichen

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Forschung

Medizin

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

The ÃƒÅ½Ã‚Â±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Die α-Test: Rapid Zellfreie CD4-Zählung mit ganzen Speichel

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

Quantitative massenspektrometrische Analyse von Krebs-Zellen-Proteome Abgeleitet von Liquid und soliden Tumoren

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

Präklinische Orthotopische Mausmodell der menschlichen Prostata-Krebs

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Ein detailliertes Protokoll für Transpiration Überwachung eines neuartigen Verwendung, Klein, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

Absolute Quantification of A&#946;1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

Absolute Quantification of AÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

Absolute Quantifizierung von Aß<sub&gt; 1-42</sub&gt; In CSF eine massenspektrometrische Referenzmessverfahren verwenden

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Eine neue klinische Grade Isolationsmethode für menschliche Niere perivaskuläre Stromazellen Zellen

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

In Vitro und In Vivo Nachweis von Mitophagy in menschlichen Zellen, C. Elegansund Mäuse

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

Chirurgische Ausbildung für die Implantation der neokortikalen Mikroelektrode Arrays mit einem Formaldehyd-feste menschliche Kadaver-Modell

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...