Name: optimal preparation of formalin fixed samples for peptide based matrix assisted laser desorption/ionization mass spectrometry imaging workflows
Uploaded: 2018-01-16T14:00:00
Description: Watch this Scientific Journal Video about Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows at JoVE.com

Författarna vill erkänna Sydney Medical School Foundation och Blues och Stiftelsen för att finansiera en del av detta arbete genom deras PhD scholarship program för Alzheimers Sjukdomforskning och en ARC Discovery bidrag (DP160102063) till PKW.

Varning: Alla tillämpliga säkerhetsåtgärder bör följas när du utför detta förfarande, inklusive användning av lämplig personlig skyddsutrustning (PPE) (t.ex. lab rockar, nitrilhandskar, skyddsglasögon, etc.)
1. beredning av reagens och utrustning
<ol><li>
Beredning av lösningar
	<ol>
<li>För att förbereda 500 mL Carnoys vätska, blanda 100% etanol (EtOH), kloroform och koncentrerad ättiksyra i en 6: förhållandet 3:1 v/v/v i en ren glasflas...

<ol>
	<li>Schwartz, S. A., Reyzer, M. L., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tissue+analysis+using+matrix-assisted+laser+desorption/ionization+mass+spectrometry:+practical+aspects+of+sample+preparation.">Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation.</a> J Mass Spectrom. 38 (7), 699-708 (2003).</li><li>Caprioli, R. M., Farmer, T. B., Gile, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Biological+Samples:+Localization+of+Peptides+and+Proteins+Using+MALDI-TOF+MS.">Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI-TOF MS.</a> Anal Chem. 69 (23), 4751-4760 (1997).</li><li>Jackson, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-Ion+Mobility+Mass+Spectrometry+of+Lipids+in+Negative+Ion+Mode.">MALDI-Ion Mobility Mass Spectrometry of Lipids in Negative Ion Mode.</a> Analytical methods : advancing methods and applications. 6 (14), 5001-5007 (2014).</li><li>O'Rourke, M. B., Djordjevic, S. P., Padula, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-instrument-based+method+for+the+analysis+of+formalin-fixed+paraffin-embedded+human+spinal+cord+via+matrix-assisted+laser+desorption/ionisation+imaging+mass+spectrometry.">A non-instrument-based method for the analysis of formalin-fixed paraffin-embedded human spinal cord via matrix-assisted laser desorption/ionisation imaging mass spectrometry.</a> Rapid Commun Mass Spectrom. 29 (19), 1836-1840 (2015).</li><li>O'Rourke, M. B., Raymond, B. B. A., Djordjevic, S. P., Padula, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+cost-effective+method+for+the+analysis+of+fresh+frozen+tissue+sections+via+matrix-assisted+laser+desorption/ionisation+imaging+mass+spectrometry.">A versatile cost-effective method for the analysis of fresh frozen tissue sections via matrix-assisted laser desorption/ionisation imaging mass spectrometry.</a> Rapid Commun Mass Spectrom. 29 (7), 637-644 (2015).</li><li>Chughtai, K., Heeren, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+imaging+for+biomedical+tissue+analysis.">Mass spectrometric imaging for biomedical tissue analysis.</a> Chem Rev. 110 (5), 3237-3277 (2010).</li><li>Ye, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+mass+spectrometry-assisted+molecular+imaging+of+metabolites+during+nitrogen+fixation+in+the+Medicago+truncatula-Sinorhizobium+meliloti+symbiosis.">MALDI mass spectrometry-assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula-Sinorhizobium meliloti symbiosis.</a> Plant J. 75 (1), 130-145 (2013).</li><li>Powers, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+imaging+mass+spectrometry+profiling+of+N-glycans+in+formalin-fixed+paraffin+embedded+clinical+tissue+blocks+and+tissue+microarrays.">MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.</a> PLoS One. 9 (9), 10655 (2014).</li><li>Rompp, A., Spengler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging+with+high+resolution+in+mass+and+space.">Mass spectrometry imaging with high resolution in mass and space.</a> Histochem Cell Biol. 139 (6), 759-783 (2013).</li><li>Shariatgorji, M., Svenningsson, P., Andren, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging,+an+emerging+technology+in+neuropsychopharmacology.">Mass spectrometry imaging, an emerging technology in neuropsychopharmacology.</a> Neuropsychopharmacology. 39 (1), 34-49 (2014).</li><li>Alexandrov, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+imaging+mass+spectrometry:+statistical+data+analysis+and+current+computational+challenges.">MALDI imaging mass spectrometry: statistical data analysis and current computational challenges.</a> BMC Bioinformatics. 13, 11 (2012).</li><li>Weaver, E. M., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mass+spectrometry:+From+tissue+sections+to+cell+cultures.">Imaging mass spectrometry: From tissue sections to cell cultures.</a> Adv Drug Deliv Rev. 65 (8), 1039-1055 (2013).</li><li>Watrous, J. D., Dorrestein, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mass+spectrometry+in+microbiology.">Imaging mass spectrometry in microbiology.</a> Nat Rev Microbiol. 9 (9), 683-694 (2011).</li><li>O'Rourke, M., Padula, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Non-Instrument+Based+Preparation+of+Tissue+Samples+Destined+for+Imaging+Mass+Spectrometry+(IMS)+Analysis.">The Non-Instrument Based Preparation of Tissue Samples Destined for Imaging Mass Spectrometry (IMS) Analysis.</a> Protocol Exchange. , (2017).</li><li>O'Rourke, M. B., Padula, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+standard+of+visual+data+representation+for+imaging+mass+spectrometry.">A new standard of visual data representation for imaging mass spectrometry.</a> Proteomics Clin Appl. 11 (3-4), (2017).</li><li>O'Rourke, M. B., Padula, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+formalin-fixed,+paraffin-embedded+(FFPE)+tissue+via+proteomic+techniques+and+misconceptions+of+antigen+retrieval.">Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval.</a> Biotechniques. 60 (5), 229-238 (2016).</li><li>Casadonte, R., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+formalin-fixed+paraffin-embedded+tissue+by+MALDI+imaging+mass+spectrometry.">Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry.</a> Nat. Protocols. 6 (11), 1695-1709 (2011).</li><li>Ong, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry+Imaging+and+Identification+of+Peptides+Associated+with+Cephalic+Ganglia+Regeneration+in+Schmidtea+mediterranea.">Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea.</a> J Biol Chem. 291 (15), 8109-8120 (2016).</li><li>Seeley, E. H., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+proteins+in+tissues+by+mass+spectrometry.">Molecular imaging of proteins in tissues by mass spectrometry.</a> Proc Natl Acad Sci U S A. 105 (47), 18126-18131 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-smear+showing+Experimental+Infection+with+Herpetomonas.">Blood-smear showing Experimental Infection with Herpetomonas.</a> Proc R Soc Med. 18, 55 (1925).</li><li>Gorrie, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+human+OEC-derived+cell+transplants+in+rodent+spinal+cord+contusion+injury.">Effects of human OEC-derived cell transplants in rodent spinal cord contusion injury.</a> Brain Res. 1337, 8-20 (2010).</li><li>Wu, Q., Comi, T. J., Li, B., Rubakhin, S. S., Sweedler, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-Tissue+Derivatization+via+Electrospray+Deposition+for+Matrix-Assisted+Laser+Desorption/Ionization+Mass+Spectrometry+Imaging+of+Endogenous+Fatty+Acids+in+Rat+Brain+Tissues.">On-Tissue Derivatization via Electrospray Deposition for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Endogenous Fatty Acids in Rat Brain Tissues.</a> Anal Chem. 88 (11), 5988-5995 (2016).</li><li>Sturm, R. M., Greer, T., Chen, R., Hensen, B., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+NIMS+and+MALDI+platforms+for+neuropeptide+and+lipid+mass+spectrometric+imaging+in+C.+borealis+brain+tissue.">Comparison of NIMS and MALDI platforms for neuropeptide and lipid mass spectrometric imaging in C. borealis brain tissue.</a> Anal Methods. 5 (6), 1623-1628 (2013).</li><li>Kompauer, M., Heiles, S., Spengler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+pressure+MALDI+mass+spectrometry+imaging+of+tissues+and+cells+at+1.4-mum+lateral+resolution.">Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-mum lateral resolution.</a> Nat Methods. 14 (1), 90-96 (2017).</li><li>Yang, J., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+sublimation/recrystallization+for+imaging+proteins+by+mass+spectrometry+at+high+spatial+resolution.">Matrix sublimation/recrystallization for imaging proteins by mass spectrometry at high spatial resolution.</a> Anal Chem. 83 (14), 5728-5734 (2011).</li><li>Rohner, T. C., Staab, D., Stoeckli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+mass+spectrometric+imaging+of+biological+tissue+sections.">MALDI mass spectrometric imaging of biological tissue sections.</a> Mech Ageing Dev. 126 (1), 177-185 (2005).</li><li>Li, B., Bhandari, D. R., Rompp, A., Spengler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+MALDI+mass+spectrometry+imaging+of+gallotannins+and+monoterpene+glucosides+in+the+root+of+Paeonia+lactiflora.">High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora.</a> Sci Rep. 6, 36074 (2016).</li><li>Spengler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging+of+biomolecular+information.">Mass spectrometry imaging of biomolecular information.</a> Anal Chem. 87 (1), 64-82 (2015).</li><li>Widlak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+molecular+signatures+of+oral+squamous+cell+carcinoma+and+normal+epithelium+-+application+of+a+novel+methodology+for+unsupervised+segmentation+of+imaging+mass+spectrometry+data.">Detection of molecular signatures of oral squamous cell carcinoma and normal epithelium - application of a novel methodology for unsupervised segmentation of imaging mass spectrometry data.</a> Proteomics. 16 (11-12), 1613-1621 (2016).</li></ol>

Detta protokoll var utformad för att maximera generationen av joniseringsbara molekylstorleken samtidigt eliminera omlokaliseringar av analyter. De viktigaste faktorerna involverar använder samma tvingande principen vid applicering matris, smälta provet eller recrystallizing efter sublimering24; nämligen, att en även nedfall av ånga, matris eller annars måste skapas och underhållas. Pipettering lösningsmedel tvättar för omkristallisering och matsmältning, jämnt under provet, förhindr...

Matrix-assisted laser desorption/jonisering masspektrometri imaging (MALDI MSI) har varit anställd som en bild baserad teknik för två decennier1,2, analysera en rad biomolekyler inklusive: lipider3, peptider2 ,4, proteiner2,5, metaboliter6,7, N-glycans8och syntetiska molekyler såsom terapeutiska läkemedel9,10. Antalet publikationer visar nyttan av denna teknik har vuxit betydligt under de senaste decenniet6,11,12,13. Vissa molekyler, såsom lipider, är relativt lätt att analysera via MALDI MSI, som de jonisera lätt på grund av sin kemiska natur och därmed kräver lite tidigare beredning3. Men för svårare mål såsom peptider är stegen som krävs för att effektivt jonisera dessa molekyler omfattande och allmänt komplicerade14. Närvarande finns det mycket få publikationer som syftar till att adress eller påvisa reproducerbarhet i de metoder som används för att förbereda vävnad för denna unika visuella teknik15. Därför har vi sammanställt observationer och genomfört optimeringar till en enda, enkel att genomföra, metodik som bör kräva lite till ingen modifiering, för analys av peptider från en formaldehyd tvärbunden vävnad källa14.
I detta manuskript, har vi beskrivit en validerad, låg kostnad reproducerbar metod för detektering och spatial kartläggning av peptider, genereras från formalin-fasta frysta (FFF) och formalin-fast paraffin-inbäddat (FFPE) vävnadssnitt. Denna metod inte kräver eller förlita sig på någon specialiserad instrumentation3. Specifikt, vi tar itu med de många aspekterna av specialiserade provberedning nödvändigt att analysera peptider; steg som antigen retrieval16 och matrix beläggning. Våra protokoll använder också billig utrustning och reagenser, vilket gör denna metod tillgänglig för en bredare gemenskap som annars inte har råd den alternativa robotic apparater17.
Resonemanget bakom utveckla en manuell prov förberedelse metod var tvåfaldigt: för det första användningen av en sublimator skapar en konsekvent och homogen beläggning av matrix kristaller som är ~ 1 µm i längd18, något ouppnåeligt med vanligare sprutning tekniker. För det andra, relativt litet Ställ kostnader: den totala kostnaden av anpassade apparater var <$ 1500 AUD. Vi noterar när det gäller kostnadseffektivitet, priset per prov är långt billigare när det finns inga automatiska maskiner inblandade. Användning av sublimering har rapporterats tidigare, men bäst av vår kunskap, steg för steg metoder som beskriver denna process och provberedning inte har rapporterats eller beskrivna i litteraturen.
Detta protokoll syftar till att bistå forskare som har tillgång till en MALDI masspektrometer och som är inställda på att generera rumslig information i förhållande till en bio-molekyl intresse19. I huvudsak är MALDI MSI en form av histologiska screening som inte bygger på antikroppar eller fläckar2.

<table border="0" cellpadding="0" cellspacing="0" style="width:820px;" width="820">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryo Microtome</td>
			<td style="width:121px;">Leica</td>
			<td style="width:160px;">CM3050</td>
			<td style="width:275px;">For preparation and section of tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Indium Tin Oxide Microscope slides</td>
			<td style="width:121px;">Bruker</td>
			<td>8237001</td>
			<td>For preparation and section of tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Coplin Jars</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>S5516</td>
			<td>For preparation and section of tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Pressure Cooker</td>
			<td style="width:121px;">Kambrook</td>
			<td>KPR620BSS</td>
			<td>For preparation and section of tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sublimator</td>
			<td style="width:121px;">Chem Glass</td>
			<td>NA</td>
			<td>For sublimation procedure. Similar in design to the CG-3038 however it was custom made&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sand bath</td>
			<td style="width:121px;">NA</td>
			<td>NA</td>
			<td>For sublimation procedure. Fine grade river sand held in folded aluminium foil sourced from outside not from any specific company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Glass Petri Dish</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>CLS70165100</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Vacuum Pump</td>
			<td style="width:121px;">NA</td>
			<td>NA</td>
			<td>For sublimation procedure. Sourced as a spare part from an old mass spectrometer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cold trap</td>
			<td style="width:121px;">Chem Glass</td>
			<td>CG-4510-02</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Hot Plate</td>
			<td style="width:121px;">John Morris</td>
			<td>EW-15956-32.&#160;</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Plastic petri dish</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>Z717223</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">37 &#176;C incubator</td>
			<td style="width:121px;">NA</td>
			<td>NA</td>
			<td>For sublimation procedure. Not applicable, incubator is non sterile and over 30 years old&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Blotting paper</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>P7796</td>
			<td>For sublimation procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Nitrocellulose&#160;</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>N8395</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Acetone</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>650501</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Xylene</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>214736</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">100% EtOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>1.02428</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">70% EtOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>NA</td>
			<td>For washing of slides. Made in lab from 95% stock ethanol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Chloroform</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>C2432</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Glacial Acetic Acid</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>ARK2183</td>
			<td>For washing of slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Tris HCL pH 8.8</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>TRIS-RO</td>
			<td>For proteolytic cleavage. Powder made to 1M followed by equilibration with 32% HCl to PH 8.8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Milli Q Ultra-Pure Water</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>NA</td>
			<td>For proteolytic cleavage. Purification performed in house by sartorious water purification system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Ammonium Bircarbonate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>A6141</td>
			<td>For proteolytic cleavage.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Trypsin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>T0303</td>
			<td>For proteolytic cleavage.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CHCA Matrix</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>C2020</td>
			<td>For recrystallisation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Acetonitrile</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>1.00029</td>
			<td>For recrystallisation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Trifluoroacetic Acid (TFA)&#160;</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>302031</td>
			<td>For recrystallisation.</td>
		</tr>
	</tbody>
</table>

optimal preparation of formalin fixed samples for peptide based matrix assisted laser desorption/ionization mass spectrometry imaging workflows

Användning av matrix-assisted laser desorption/jonisering, masspektrometri imaging (MALDI MSI) har snabbt expanderat, eftersom denna teknik analyserar en värd av biomolekyler från droger och lipider till N-glycans. Även om olika provberedningstekniker finns, förblir att upptäcka peptider från formaldehyd bevarade vävnader en av de svåraste utmaningarna för denna typ av massa spektrometriska analys. Därför har vi skapat och optimerat en robust metod som bevarar den rumsliga informationen i provet, medan framkalla det största antalet joniseringsbara peptider. Vi har också syftar till att uppnå detta på ett enkelt och kostnadseffektivt sätt, vilket eliminerar potentiella bias eller beredning fel, som kan uppstå när du använder automatisk instrumentering. Slutresultatet är ett reproducerbart och billig protokoll.

Om följt korrekt, producerar detta protokoll bilder som tydligt representerar brutto morfologi av vävnad utan några repor eller andra mindre deformationer (figur 1). Den idealiska valideringen för ett korrekt utfört provberedning, är förmågan att skilja mellan olika fysiska strukturer genom att ändra den molekyl som tittade på (figur 2).
En bra guide för att ...

Watch this Scientific Journal Video about Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows at JoVE.com

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

Optimal förberedelse av formalinbeständiga prover för peptid baserat Matrix assisterad Laser Desorption/jonisering masspektrometri Imaging arbetsflöden

Det här protokollet beskriver en reproducerbar och pålitlig metod för sublimering-baserade beredning av formalinbeständiga vävnad avsedd för imaging masspektrometri.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Research

JoVE Journal

Biochemistry

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless Capillary ElectrophoresisÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless kapillärelektrofores-masspektrometri för Metabolic Profiling av biologiska prover

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Tidsupplöst elektrosprayjonisering Väte-deuterium Exchange-masspektrometri för att studera Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Lösa affinitetsrenat proteinkomplex av Blue Native PAGE och protein Korrelation profilering

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Glycoproteomics av ​​den extracellulära matrisen: En metod för intakt glykopeptiden analys med masspektrometri

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Enkelt att använda arkitekturer i Protein-baserade hydrogeler för Cell kultur applikationer

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Provberedning för Mass Spectrometry-baserade identifiering av RNA-bindande regioner

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Detektion av protein ubiquitination platser av peptidanrikning och masspektrometri

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Ett plasmaprovförberedelse för masspektrometri med hjälp av en automatiserad arbetsstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Provberedning för probe electrospray jonisering Masspektrometri

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Kvantitativ analys av cellulär lipidom e av Saccharomyces Cerevisiae med hjälp av flytande kromatografi tillsammans med Tandem Mass Spectrometri

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...