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

Dit werk werd ondersteund door de National Institutes of Health Grants (R01-AI067395-01, R21-R022754-01 en R21-I58002-01). Wij danken C. Niemeyer voor de kritische lezing van het manuscript.

1. Oprichting van LLUF Probes <ol><li> De polyethersulfone membranen (2 cm 2) werden afgesloten met driehoek polypropyleen paddles (University of California, San Diego) door lijmen membranen met epoxy op de grens van peddels. Een negatief geladen polyethersulfon membraan met een molecuulgewicht drempelwaarde (MWCO) op 30 kDa was gebruikt. </li><li> Een teflon gefluoreerde etheen-propeen buis (inwendige diameter / buitendiameter 0.35/0.50 cm) werd aan een cilinder uitgang van een driehoek polypropyleen paddl...

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Wij hebben gevonden dat veel peptide fragmenten bestaan ​​menselijke onverteerd speeksel. Deze peptide fragmenten daarvan aan verschillende vormen van proline-rijke eiwitten, actine, alfa-amylase, alpha 1 globine, beta-globine, histain 1, keratine 1, mucine 7 polymère immunoglobuline receptor, satherin, S100A9. Er kunnen vele factoren de productie van peptiden met een onbepaald knipplaatsen. Bijvoorbeeld, sommige peptidefragmenten van nature aanwezig in humaan speeksel. Veel peptiden met-PQ C-termini vastgesteld <s...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Reacties </td></tr><tr><td> Polyethersulfon membranen </td><td> Pall Corporation </td><td></td><td> 30 kDa MWCO </td></tr><tr><td> Teflon gefluoreerde etheen-propeen buis </td><td> Upchurch Wetenschappelijk </td><td></td><td></td></tr><tr><td> Blue dextran </td><td> Sigma </td><td></td><td></td></tr><tr><td> Nano LC systeem </td><td> Eksigent </td><td></td><td></td></tr><tr><td> C18 val kolom </td><td> Agilent </td><td> 5065-9913 </td><td></td></tr><tr><td> LTQ lineaire ion-trap massaspectrometer </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% mierenzuur </td><td> JT Baker </td><td> 9832-03 </td><td> LC / MS klasse </td></tr><tr><td> Water-0.1% mierenzuur </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

Hoewel het menselijk speeksel proteoom en peptidoom zijn geopenbaard 1-2 werden ze majorly geïdentificeerd uit eiwit hydrolysaten van speeksel eiwitten. Identificatie van de inheemse peptidoom van het menselijk speeksel zonder voorafgaande vertering met exogene enzymen wordt het absoluut noodzakelijk, omdat natieve peptiden in menselijk speeksel verstrekt potentiële waarden voor de diagnose van de ziekte, het voorspellen van progressie van de ziekte, en het toezicht op de therapeutische werkzaamheid. Passende steekproef is een cruciale stap voor de verbetering van de identificatie van menselijke inheemse speeksel peptidoom. Traditionele methoden van bemonstering van menselijk speeksel met centrifugeren om vuil te verwijderen 3-4 mei te veel tijd in beslag van toepassing te zijn voor klinisch gebruik. Bovendien kan vuil verwijderd door centrifugatie niet kunnen meeste geïnfecteerde pathogenen reinigen en de hoge overvloed eiwitten die vaak belemmeren de identificatie van geringe hoeveelheden peptidoom verwijderen. Conventionele proteomische benaderingen die priMarily gebruik tweedimensionale gel-elektroforese (2-DE) gels in conjugatie met in-gel digestie staat zijn om vele speeksel eiwitten 5-6. Deze aanpak is algemeen niet gevoelig genoeg om geringe hoeveelheden peptiden / eiwitten te detecteren. Vloeistofchromatografie-massaspectrometrie (LC-MS) op basis van proteomics is een alternatief dat eiwitten zich kunnen identificeren zonder voorafgaande 2-DE-scheiding. Hoewel deze benadering levert een hogere gevoeligheid moet algemeen vooraf mon-fractionering 7 en pre-digestie met trypsine, waardoor het moeilijk voor klinisch gebruik. Om de hinder in de massa spectrometrie als gevolg van monstervoorbereiding te omzeilen, hebben we een techniek genaamd capillaire ultrafiltratie (CUF) sondes 8-11. De gegevens van ons laboratorium aangetoond dat de CUF probes in staat zijn om het vastleggen van eiwitten in vivo van verschillende micro-omgevingen bij dieren in een dynamische en minimaal invasieve manier 8 -11. Geen centrifugeren is nodig omdat een negatieve druk wordt gecreëerd door gewoon spuit te trekken tijdens de monstername. De CUF sondes in combinatie met LC-MS hebben met succes geïdentificeerd tryptische-eiwitten verteerd 8-11. In deze studie hebben we een upgrade van de ultrafiltratie sampling techniek door het creëren van een lolly-achtige ultrafiltratie (LLUF) probe die gemakkelijk kunnen passen in de menselijke mondholte. De directe analyse van LC-MS zonder trypsinedigestie gebleken dat menselijk speeksel aangekocht bevat veel peptide fragmenten afkomstig van verschillende eiwitten. Bemonstering speeksel met LLUF sondes vermeden centrifugeren, maar effectief verwijderd veel groter en hoge overvloed eiwitten. Onze massaspectrometrische resultaten blijkt dat veel lage abundantie peptiden werden gedetecteerd na het uitfilteren van grotere eiwitten met LLUF sondes. Detectie van lage abundantie heeft speeksel peptiden was onafhankelijk van de verschillende voor-stap voorbeeld scheiding met chromatografie. Voor klinische toepassing, de LLUF sondes te nemend met LC-MS zou kunnen worden gebruikt in de toekomst progressie van de ziekte van speeksel te controleren.

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

Rekening houdend met speeksel bemonstering voor toekomstige klinische toepassing, is een lolly-achtige ultrafiltratie (LLUF) probe gemaakt om te passen in de menselijke mondholte. Directe analyse van onverteerd speeksel door nanoLC-LTQ massaspectrometrie werd aangetoond dat LLUF sondes om grote eiwitten en een hoge overvloed eiwitten te verwijderen, en maak een lage overvloedige peptiden meer aantoonbaar.

Bemonstering Human Inheemse Speeksel peptidoom Met behulp van een Lollipop-Like Ultrafiltratie Probe: vereenvoudigen en te verbeteren Peptide Detection voor Klinische massaspectrometrie

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

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Met behulp van visuele en narratieve methoden om een ​​eerlijk proces te bereiken in de klinische zorg

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

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

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

De α-test: Rapid Celvrije CD4 Enumeration met hele Speeksel

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

Het verzamelen van speeksel en meten cortisol en Alpha-amylase in Frail Gemeenschap wonende ouderen via mantelzorgers

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

Kwantitatieve Massaspectrometrische Profilering van Kanker-cel proteoom Afgeleid Van vloeibare en vaste 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

Pre-klinische Orthotope muizenmodel van Human prostaatkanker

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

Een gedetailleerd protocol voor het Zweet Monitoring Met behulp van een roman, Kleine, draadloze apparaten

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 kwantificering van Aß<sub&gt; 1-42</sub&gt; In CSF met behulp van een Massaspectrometrische Reference Measurement Procedure

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

Een nieuwe klinische Grade isolatie methode voor menselijke nieren gerelateerde stromale cellen

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 en In Vivo opsporing van Mitophagy in menselijke cellen, C. Elegansen muizen

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 opleiding voor de inplanting van Neocortical micro-elektrode Arrays gebruikmakend van een menselijk kadaver Formaldehyde-fixed-Model

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