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 arbeidet ble støttet av National Institutes of Health Grants (R01-AI067395-01, R21-R022754-01, og R21-I58002-01). Vi takker C. Niemeyer for kritisk lesing av manuskriptet.

1. Opprettelse av LLUF Probes <ol><li> De polyetersulfon membraner (2 cm 2) ble forseglet med trekant polypropylen padleårer (University of California, San Diego) som limes membraner med epoxy på grensene til padleårer. Et negativt ladet polyetersulfon membran med en molekylvekt cut-off (MWCO) på 30 kDa ble brukt. </li><li> En teflon fluorinert etylen propylen tube (indre diameter / ytre diameter, 0.35/0.50 cm) ble festet til en sylinder exit av en trekant polypropylen padle så LLUF sonden kan kobles t...

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Vi har funnet at mange peptidfragmenter eksisterer i menneskets ufordøyd spytt. Disse peptidfragmenter er derivater fra ulike former for prolin-rike proteiner, aktin, alfa amylase, alpha 1 globin, beta globin, histain 1, keratin 1, mucin 7, polymeriske immunglobulin reseptoren, satherin, S100A9. Det kan være mange faktorer som bidrar til produksjon av peptider med undetermined kutteseter. For eksempel kan noen peptidfragmenter være naturlig tilstede i menneskelig helhet spytt. Mange peptider med-PQ C-Termini ble iden...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Polyetersulfon membraner </td><td> Pall Corporation </td><td></td><td> 30 kDa MWCO </td></tr><tr><td> Teflon fluorinert etylen propylen tube </td><td> Upchurch Scientific </td><td></td><td></td></tr><tr><td> Blå dekstran </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 felle kolonne </td><td> Agilent </td><td> 5065-9913 </td><td></td></tr><tr><td> LTQ lineær ion-felle 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> Acetonitrile-0,1% maursyre </td><td> JT Baker </td><td> 9832-03 </td><td> LC / MS karakter </td></tr><tr><td> Vann-0,1% maursyre </td><td> JT Baker </td><td> 9834-03 </td><td> LC / MS karakter </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 om menneskelige spytt proteomet og peptidome har blitt avslørt 1-2 de ble majorly identifisert fra tryptic oppsummeringer av spytt proteiner. Identifisering av urfolk peptidome av menneskers spytt uten forutgående fordøyelsen med eksogene enzymer blir viktig, ettersom innfødte peptider i human saliva gi potensielle verdier for diagnostisering sykdom, forutsi sykdomsutvikling, og overvåking terapeutisk effekt. Egnet prøvetaking er et kritisk punkt for forbedring av identifisering av menneskelig innfødte spytt peptidome. Tradisjonelle metoder for prøvetaking menneskelig spytt involverer sentrifugering for å fjerne rusk 3-4 kan være for tidkrevende å være aktuelt for klinisk bruk. Videre kan rusk fjerning ved sentrifugering være ute av stand til å rense de fleste av de smittede patogener og fjern de høye overflod proteiner som ofte hindrer identifisering av lav overflod peptidome. Konvensjonelle proteomikk tilnærminger som prifremst utnytte to-dimensjonal elektroforese (2-DE) gels i konjugering med i-gel fordøyelse er i stand til å identifisere mange spytt proteiner 5-6. Imidlertid er denne tilnærmingen som regel ikke tilstrekkelig følsom for å oppdage lav overflod peptider / proteiner. Væskekromatografi-massespektrometri (LC-MS) baserte proteomikk er et alternativ som kan identifisere proteiner uten forutgående to-DE separasjon. Selv om denne tilnærmingen gir høyere følsomhet, må det vanligvis før prøven før fraksjonering 7 og pre-fordøyelse med trypsin, som gjør det vanskelig for klinisk bruk. For å omgå hindringen i massespektrometri grunn prøveopparbeidelse, har vi utviklet en teknikk som kalles kapillær ultrafiltrasjon (CUF) sonder 8-11. Data fra vårt laboratorium viste at CUF sondene er i stand til å fange proteiner in vivo fra ulike microenvironments i dyr i en dynamisk og minimalt invasiv måte 8 -11. Ingen sentrifugering er nødvendig fordi et undertrykk blir skapt ved å sprøyte trekke under prøvetakingen. De CUF prober kombinert med LC-MS har blitt identifisert tryptic-fordøyde proteiner 8-11. I denne studien, oppgradert vi ultrafiltrasjon prøvetaking teknikken ved å opprette en lollipop-lignende ultrafiltrasjon (LLUF) sonde som lett kan passe i den menneskelige munnhulen. Den direkte analyse av LC-MS uten trypsin fordøyelsen viste at menneskelige spytt indigenously inneholder mange peptidfragmenter fra ulike proteiner. Prøvetaking spytt med LLUF sonder unngått sentrifugering men effektivt fjernet mange større og høy individtetthet proteiner. Våre massespektrometrisk resultater vist at mange lav overflod peptider ble påvisbar etter å filtrere ut større proteiner med LLUF sonder. Påvisning av lav overflod spytt peptider var uavhengig av flere trinn prøve separasjon med kromatografi. For klinisk anvendelse, de LLUF sonder innlemmed med LC-MS kan potensielt brukes i fremtiden for å overvåke sykdomsutvikling fra spytt.

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

Vurderer spytt prøvetaking for fremtidig klinisk anvendelse, ble en lollipop-lignende ultrafiltrasjon (LLUF) sonde fabrikert for å passe i den menneskelige munnhulen. Direkte analyse av ufordøyd spytt ved NanoLC-LTQ massespektrometri demonstrert evne LLUF sonder for å fjerne store proteiner og høye overflod proteiner, og gjør lite tallrike peptider mer synlig.

Prøvetaking Menneskelig Indigenous Spytt Peptidome Bruke en Lollipop-Like ultrafiltrasjon Probe: Forenkle og forbedre Peptide 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 ÃƒÅ½Ã‚Â±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Den α-test: Rapid Cell-free CD4 Enumeration Bruke Hele Spytt

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 av kreft-celle proteom Derived From Flytende og solide 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

Preklinisk ortotopiske Murine Model of Human prostatakreft

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 detaljert protokoll for Svette Monitoring Ved hjelp av en roman, Small, 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

Absolutt Kvantifisering av Ap<sub&gt; 1-42</sub&gt; I CSF Ved hjelp av en massespektrometrisk Reference målingsprosedyre

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

Romanen klinisk Grade isolasjon metode for menneskelig Perivascular Stromal nyreceller

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 i Vivo oppdagelse av Mitophagy i menneskelige 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

Kirurgisk opplæring for implantering av Neocortical Microelectrode matriser med formaldehyd-fast menneskelige Cadaver modell

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