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

Dette arbejde blev støttet af National Institutes of Health Tilskud (R01-AI067395-01, R21-R022754-01, og R21-I58002-01). Vi takker C. Niemeyer til kritisk læsning af manuskriptet.

1. Oprettelse af LLUF Probes <ol><li> De polyethersulfon membraner (2 cm 2) blev forseglet med trekant polypropylen padler (University of California, San Diego), ved at lime membraner med epoxy på grænsen af padler. En negativt ladet polyethersulfon-membran med en molekylvægt cut-off (MWCO) på 30 kDa blev anvendt. </li><li> En teflon fluoreret ethylenpropylen rør (indre diameter / ydre diameter, 0.35/0.50 cm) blev fastgjort til en cylinder udgang af en trekant polypropylen skovl så LLUF proben kan for...

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Vi har fundet, at mange peptidfragmenter findes i humant ufordøjet spyt. Disse peptidfragmenter er derivater fra forskellige former for prolin-rige proteiner, actin, alfa-amylase, alfa 1 globin, beta-globin, histain 1, keratin 1, mucin 7, polymert immunoglobulin receptor, satherin, S100A9. Der kan være mange faktorer, der bidrager til produktion af peptider med ubestemte spaltningssteder. For eksempel kan nogle peptidfragmenter være naturligt til stede i humant spyt. Mange peptider med-PQ C-termini, blev identificere...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nr. </td><td> Kommentarer </td></tr><tr><td> Polyethersulfon membraner </td><td> Pall Corporation </td><td></td><td> 30 kDa MWCO </td></tr><tr><td> Teflon fluoreret ethylenpropylen rør </td><td> Upchurch Scientific </td><td></td><td></td></tr><tr><td> Blue dextran </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 fælde kolonne </td><td> Agilent </td><td> 5065-9913 </td><td></td></tr><tr><td> LTQ lineær ion-fælde massespektrometer </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% myresyre </td><td> JT Baker </td><td> 9832-03 </td><td> LC / MS grad </td></tr><tr><td> Vand 0,1% myresyre </td><td> JT Baker </td><td> 9834-03 </td><td> LC / MS grad </td></tr></tbody></table>

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

Selv humant spyt proteom og peptidome er blevet afsløret 1-2 blev de majorly identificeret fra tryptiske fordøjelser af spyt-proteiner. Identifikation af naturligt forekommende peptidome af humant spyt uden forudgående fordøjelse med exogene enzymer bliver det nødvendigt, eftersom native peptider i humant spyt tilvejebringe potentielle værdier til diagnosticering sygdom, forudsigelse sygdomsprogression, og overvågning terapeutisk virkning. Passende stikprøve er et afgørende skridt til forbedring af identifikation af humane indfødte spyt peptidome. Traditionelle metoder til prøvetagning menneskelig spyt involverer centrifugering for at fjerne snavs 3-4 kan være for tidskrævende at være gældende for klinisk brug. Endvidere kan debris fjernes ved centrifugering være i stand til at rense de fleste af de inficerede patogener og fjerne de høje tæthed proteiner, ofte hindrer identifikation af lav tæthed peptidome. Konventionelle proteom tilgange, PRIprimært anvender to-dimensional gelelektroforese (2-DE) geler i konjugation med i-gel fordøjelse stand til at identificere mange spyt-proteiner 5-6. Men denne fremgangsmåde er generelt ikke tilstrækkeligt følsomme til at detektere lav hyppighed peptider / proteiner. Væskekromatografi-massespektrometri (LC-MS) baserede proteomik er et alternativ, som kan identificere proteiner uden 2-DE separation. Selvom denne fremgangsmåde giver en højere følsomhed, er det generelt nødvendigt forud prøve præ-fraktionering 7 og præ-fordøjelse med trypsin, hvilket gør det vanskeligt for klinisk anvendelse. For at omgå den hindring i massespektrometri på grund af prøveforberedelse, har vi udviklet en teknik kaldet kapillær ultrafiltrering (CUF) sonder 8-11. Data fra vores laboratorium vist, at de CUF prober er i stand til at opfange proteiner in vivo af forskellige mikromiljøer i dyr i en dynamisk og minimalt invasiv måde 8 -11. Ingen centrifugering er nødvendig, eftersom et negativt tryk skabes ved at sprøjte trække under prøveopsamling. De CUF prober kombineret med LC-MS succes har identificeret tryptisk-fordøjede proteiner 8-11. I denne undersøgelse har vi opgraderet ultrafiltreringen stikprøveudtagningsteknik ved at skabe en slikkepind-lignende ultrafiltrering (LLUF) probe, der let kan passe ind i den humane mundhule. Den direkte analyse ved LC-MS uden trypsinfordøjelse viste, at humant spyt udvindes indeholder mange peptidfragmenter afledt af forskellige proteiner. Sampling spyt med LLUF prober undgås centrifugering men effektivt fjernes mange større og høj forekomst proteiner. Vores massespektrometriske resultater viste, at mange med lav hyppighed peptider blev spores efter filtrere større proteiner med LLUF sonder. Påvisning af lav hyppighed spyt peptider var uafhængig af multiple-trin prøve separation med kromatografi. For kliniske anvendelse, indarbejde LLUF sonderd med LC-MS potentielt kan anvendes i fremtiden til at overvåge sygdomsprogression fra spyt.

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

Betragtning spyt prøver for fremtidig klinisk anvendelse, blev en slikkepind-lignende ultrafiltrering (LLUF) probe fremstillet til at passe i den humane mundhule. Direkte analyse af ufordøjet spyt ved NanoLC-LTQ massespektrometri viste evnen hos LLUF prober til at fjerne store proteiner og høje tæthed proteiner, og med en lav overflod peptider mere detekterbar.

Prøveudtagning Menneskelig Indigenous Spyt Peptidome Brug en Lollipop-Like Ultrafiltrering Probe: forenkle og forbedre Peptid Detection for Klinisk massespektrometri

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

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

Den α-test: Rapid Cellefri CD4 Optælling Anvendelse helspyt

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

Kvantitativ Massespektrometrisk profilering kræft-celle proteomer arvet fra flydende og faste tumorer

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

Prækliniske ortotopisk Murine Model of Human Prostata Cancer

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

En detaljeret protokol for Sved Overvågning Ved hjælp af en roman, Small, trådløs enhed

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

Absolut Kvantificering af Ap<sub&gt; 1-42</sub&gt; I CSF Brug massespektrometrisk 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

En roman kliniske Grade Isolation metode for menneskelige nyre Perivascular stromale celler

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 og In Vivo påvisning af Mitophagy i humane celler, C. Elegansog mus

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

Kirurgiske uddannelse til Implantation af Neocortical mikroelektrode Arrays ved brug af en formaldehyd-fast menneskelige Kadaver Model

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