Name: Een gestroomlijnde aanpak voor proteomics op basis van massaspectrometrie met behulp van geselecteerde weefselregios (Video) | JoVE Journal
Uploaded: 2025-04-18T14:30:00.000+00:00
Duration: 9 min
Description: Ontdek een gestroomlijnde massaspectrometrie-methode voor proteomics op specifieke weefselregio's in FFPE-monsters. Deze techniek verbetert de nauwkeurigheid van eiwitanalyse en ondersteunt onderzoek naar pancreasaandoeningen.

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</ol>

<table><tbody><tr><td>0.1% FA in ACN (LC-MS grade)</td><td>Fisher Chemical</td><td>LS120-212</td><td></td></tr><tr><td>0.1% FA in Water (LC-MS grade)</td><td>Fisher Chemical</td><td>LS118-4</td><td></td></tr><tr><td>0.5M TCEP</td><td>Thermo Scientific</td><td>77720</td><td></td></tr><tr><td>10% SDS</td><td>Invitrogen</td><td>2679093</td><td></td></tr><tr><td>1M TEAB (pH 8.5)</td><td>Sigma-Aldrich</td><td>102545001</td><td></td></tr><tr><td>1M Tris-cl (pH 8.5)</td><td>BIOSOLUTION</td><td>BTO21</td><td></td></tr><tr><td>A-14C centrifuge</td><td>Satorious</td><td>167709</td><td></td></tr><tr><td>Acetone (HPLC grade)</td><td>Fisher Scientific</td><td>A949-4</td><td></td></tr><tr><td>ACN (HPLC grade)</td><td>J.T.Baker</td><td>9017-88</td><td></td></tr><tr><td>CHCl3 (HPLC grade)</td><td>Thermo Scientific</td><td>022920.k2</td><td></td></tr><tr><td>CR paper</td><td>ADVANTEC</td><td>70406001</td><td></td></tr><tr><td>DIA-NN ver 1.9&nbsp;</td><td>Open source&nbsp;</td><td>https://github.com/vdemichev/DiaNN</td><td>Proteomics Search Engine&nbsp;</td></tr><tr><td>EPOCH2 microplate reader</td><td>Agilent&nbsp;</td><td>2106208</td><td></td></tr><tr><td>Ethanol</td><td>MERCK</td><td>K50505283 836</td><td></td></tr><tr><td>FA (LC-MS grade)</td><td>Fisher Chemical</td><td>A117-50</td><td></td></tr><tr><td>Ingenuity Pathway Analysis (IPA)</td><td>QIAGEN</td><td>830018</td><td>Bioinformatics tool</td></tr><tr><td>Lyophilizer (SRF110R+vaper trap)</td><td>Thermo Scientific</td><td>SRF-110-115</td><td></td></tr><tr><td>MeOH (HPLC grade)</td><td>MERCK</td><td>UN1230</td><td></td></tr><tr><td>Microplate BCA protein Assay kit-Reducing Agent Compatible</td><td>Thermo Scientific</td><td>23252</td><td></td></tr><tr><td>MSConvert&nbsp;</td><td>Open source&nbsp;</td><td>http://proteowizard.sourceforge.net/tools.shtml</td><td>MS data transformation software</td></tr><tr><td>Orbitrap Exploris 480</td><td>Thermo Scientific</td><td>MA10813C</td><td>MS</td></tr><tr><td>PepMAP RSLC C18 separation column&nbsp;</td><td>Thermo Scientific</td><td>ES903</td><td></td></tr><tr><td>Perseus</td><td>Open source</td><td>https://cox-labs.github.io/coxdocs/perseus_instructions.html</td><td>Statistical tool</td></tr><tr><td>PIERCE chloroacetamide No-Weigh Format</td><td>Thermo Scientific</td><td>A39270</td><td></td></tr><tr><td>PIERCE Quantitative colorimetric peptide Assay</td><td>Thermo Scientific</td><td>23275</td><td></td></tr><tr><td>Plate shaker&nbsp;</td><td>Green SSeriker</td><td>VS-202D</td><td></td></tr><tr><td>Probe sonicator</td><td>VibraCellTM</td><td>VCX750</td><td></td></tr><tr><td>Protein LoBind Tube 1.5 mL</td><td>Eppendorf</td><td>22431081</td><td></td></tr><tr><td>QSP 10 &micro;L pipette Tip</td><td>Thermo Scientific</td><td>TLR102RS-Q</td><td></td></tr><tr><td>QSP 300 &micro;L pipette Tip</td><td>Thermo Scientific</td><td>TLR106RS-Q</td><td></td></tr><tr><td>Scalpel</td><td>Bard-Parker</td><td>372615</td><td></td></tr><tr><td>S-Trap: Rapid Universal MS sample Prep</td><td>PROTIFI</td><td>CO2-mini-40</td><td></td></tr><tr><td>SureSTART Vial 0.2 mL</td><td>Thermo Scientific</td><td>6pk1655</td><td></td></tr><tr><td>TFA</td><td>Sigma-Aldrich</td><td>102614284</td><td></td></tr><tr><td>ThermoMixer C</td><td>Eppendorf</td><td>5382</td><td></td></tr><tr><td>Trypsin/Lys-C (LC-MS grade)</td><td>Promega</td><td>V5073</td><td></td></tr><tr><td>Vanquish NEO</td><td>Thermo Scientific</td><td>8348249</td><td>LC</td></tr><tr><td>Water (HPLC grade)</td><td>Honeywell</td><td>AH365-4</td><td></td></tr><tr><td>Xcalibur ver 4.7</td><td>Thermo Scientific</td><td>30966</td><td>MS data acquisition software</td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>102033629</td><td></td></tr></tbody></table>

een gestroomlijnde aanpak voor proteomics op basis van massaspectrometrie met behulp van geselecteerde weefselregio's

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Een gestroomlijnde aanpak voor proteomics op basis van massaspectrometrie met behulp van geselecteerde weefselregio's

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

a-streamlined-approach-for-mass-spectrometry-based-proteomics-using

Research

JoVE Journal

Biochemistry

Biochemie

Diepgaande analyses van kankercel-proteomen vergemakkelijken de identificatie van nieuwe doelen voor geneesmiddelen en diagnostische biomarkers. We beschrijven een experimentele workflow voor kwantitatieve analyse van (fosfo-)proteomen in kankercelsubpopulaties afgeleid van vloeibare en vaste tumoren. Dit wordt bereikt door cellulaire verrijkingsstrategieën te combineren met kwantitatieve Super-SILAC-gebaseerde massaspectrometrie.

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

We presenteren een protocol om te kwantificeren nauwkeurig eiwitten met isobaar etikettering, uitgebreide fractionering, bioinformatica tools en kwaliteitscontrole stappen in combinatie met vloeibare chromatografie geïnterfacet met een hoge resolutie massaspectrometer.

Diepe Proteoom profilering door isobaar Labeling, uitgebreide vloeistofchromatografie, massaspectrometrie en Software-bijgewoonde kwantificering

Hier presenteren we een twee-dimensionale gelelektroforese (2DE) dat is gekoppeld aan massaspectrometrie (MS) te scheiden en te identificeren menselijke hypofyse adenoom weefsel Proteoom, waarin een goede en reproduceerbare 2DE patroon. Veel eiwitten worden bij elke 2DE plek waargenomen bij het analyseren van complexe kanker Proteoom met het gebruik van hoog-gevoeligheids MS.

Twee-dimensionale gelelektroforese gekoppeld aan massaspectrometrie methoden voor een analyse van menselijke hypofyse adenoom weefsel Proteoom

We presenteren een geoptimaliseerd tmt-labelprotocol (tandem mass tag) dat gedetailleerde informatie bevat voor elk van de volgende stappen: eiwitextractie, kwantificering, neerslag, spijsvertering, etikettering, indiening bij een proteomics-faciliteit en gegevensanalyses.

TMT Sample Voorbereiding voor Proteomics Facility Indiening en latere data-analyse

Het beschreven protocol biedt een geoptimaliseerde kwantitatieve proteomics-analyse van weefselmonsters met behulp van twee benaderingen: labelgebaseerde en labelvrije kwantificering. Labelgebaseerde benaderingen hebben het voordeel van een nauwkeurigere kwantificering van eiwitten, terwijl een labelvrije aanpak kosteneffectiever is en wordt gebruikt om honderden monsters van een cohort te analyseren.

Uitgebreide workflow van op massaspectrometrie gebaseerde Shotgun Proteomics van weefselmonsters

Het protocol is bedoeld om het gebruik van een triple quadrupole mass spectrometer voor Multiple Reaction Monitoring (MRM) van eiwitten uit klinische monsters in te voeren. We hebben een systematische workflow voorzien, van monstervoorbereiding tot gegevensanalyse voor klinische monsters met alle nodige voorzorgsmaatregelen die moeten worden genomen.

Kwantitatieve Proteomics Workflow met behulp van multiple reaction monitoring gebaseerde detectie van eiwitten uit menselijk hersenweefsel

Protein Arginine (R)-methylatie is een wijdverspreide posttranslationele modificatie die meerdere biologische routes reguleert. Massaspectrometrie is de beste technologie om het R-methylproteoom wereldwijd te profileren, wanneer gekoppeld aan biochemische benaderingen voor gemodificeerde peptideverrijking. De workflow die is ontworpen voor de identificatie met hoge betrouwbaarheid van globale R-methylatie in menselijke cellen wordt hier beschreven.

Een op massaspectrometrie gebaseerde proteomics-benadering voor wereldwijde en betrouwbare eiwit-R-methylatieanalyse

Sfingolipiden zijn bioactieve metabolieten met gevestigde rollen bij menselijke ziekten. Het karakteriseren van veranderingen in weefsels met massaspectrometrie kan rollen in de ziekte-etiologie onthullen of therapeutische doelen identificeren. De OCT-verbinding die wordt gebruikt voor cryopreservatie in biorepositories interfereert echter met massaspectrometrie. We schetsen methoden om sfingolipiden in menselijke weefsels ingebed in OCT te analyseren met LC-ESI-MS / MS.

Voorbereiding van menselijke weefsels ingebed in optimale snijtemperatuurverbinding voor massaspectrometrie-analyse

Op massaspectrometrie gebaseerde proteomische gegevens zijn beschikbaar in open databases en toegankelijk met behulp van gratis tools. Gezien de complexiteit van het doorzoeken en beschrijven van databases, missen veel biologen de kennis om deze datasets te gebruiken. Hier bieden we een gids over het gebruik van gratis tools voor het doorzoeken van basisproteomische gegevens.

Navigeren door de op massaspectrometrie gebaseerde proteomische gegevens met behulp van gratis computationele tools

Conformationele flexibiliteit speelt een cruciale rol in de eiwit-functie. Hierin beschrijven we het gebruik van tijdsopgeloste electrospray ionisatie massaspectrometrie gekoppeld met waterstof deuterium ruil voor het sonderen van de snelle structurele veranderingen die functie rijden besteld en ontvouwen eiwitten.

Tijdsgeresolveerde elektrosprayionisatie waterstof-deuterium Exchange Massaspectrometrie voor het bestuderen van Protein Structure en Dynamics

Ontdek een gestroomlijnde massaspectrometrie-methode voor proteomics op specifieke weefselregio's in FFPE-monsters. Deze techniek verbetert de nauwkeurigheid van eiwitanalyse en ondersteunt onderzoek naar pancreasaandoeningen.

Een gestroomlijnde aanpak voor proteomics op basis van massaspectrometrie met behulp van geselecteerde weefselregio's

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