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

The authors would like to acknowledge the Sydney Medical School Foundation and Blues and Foundation for funding part of this work through their PhD scholarship program for Alzheimer's Disease Research and an ARC Discovery grant (DP160102063) awarded to PKW.

CAUTION: All applicable safety precautions should be followed when performing this procedure, including the use of appropriate personal protective equipment (PPE) (e.g., lab coats, nitrile gloves, safety glasses, etc.)
1. Preparation of Reagents and Equipment
<ol>
	<li>Preparation of solutions
	<ol>
		<li>To prepare 500 mL of Carnoy&#39;s fluid, mix 100% ethanol (EtOH), chloroform, and glacial acetic acid in a 6:3:1 v/v/v ratio in a clean glass Schott bottl...

<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>

This protocol was designed to maximize the generation of ionizable molecular species while eliminating delocalization of analytes. The key factors involve using the same overriding principle when applying matrix, digesting the sample, or recrystallizing after sublimation24; namely, that an even deposition of vapor, matrix or otherwise, needs to be created and maintained. Pipetting solvent washes for recrystallization and digestion, evenly underneath the sample, prevents any individual area receivi...

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has been employed as an image based technique for two decades1,2, analyzing a range of biomolecules including: lipids3, peptides2,4, proteins2,5, metabolites6,7, N-glycans8, and synthetic molecules such as therapeutic drugs9,10. The number of publications demonstrating the utility of this technique have grown significantly over the last decade6,11,12,13. Certain molecules, such as lipids, are relatively easy to analyze via MALDI MSI, as they ionize readily due to their chemical nature and thus require little prior preparation3. However, for more difficult targets such as peptides, the steps required to effectively ionize these molecules are extensive and generally complicated14. There are currently very few publications that aim to address or demonstrate reproducibility in the methodologies that are employed to prepare tissue for this unique visual technique15. For this reason, we have compiled observations and implemented optimizations into a single, easy to implement, methodology that should require little to no modification, for the analysis of peptides from a formaldehyde cross-linked tissue source14.
In this manuscript, we have described a validated, low cost reproducible methodology for the detection and spatial mapping of peptides, generated from formalin-fixed frozen (FFF) and formalin-fixed paraffin-embedded (FFPE) tissue sections. This methodology does not require or rely on any specialized instrumentation3. Specifically, we address the many aspects of specialized sample preparation necessary to analyze peptides; steps such as antigen retrieval16 and matrix coating. Our protocol also utilizes inexpensive equipment and reagents, thereby making this methodology accessible to a wider community who would otherwise be unable to afford the alternate robotic apparatus17.
The reasoning behind developing a manual sample preparation method was two-fold: Firstly, the use of a sublimator creates a consistent and homogenous coating of matrix crystals that are ~1 &#181;m in length18, something unachievable with more common spraying techniques. Secondly, the relatively small set up costs: the total cost of the custom apparatus was &#60;$1500 AUD. We note, in terms of cost effectiveness, the price per sample is far cheaper when there is no robotic machinery involved. The use of sublimation has been reported previously, however, to the best of our knowledge, step-by-step methodologies that describe this process and sample preparation have not been reported nor described in the literature.
This protocol is intended to assist researchers that have access to a MALDI mass spectrometer and who are intent on generating spatial information in relation to a bio-molecule of interest19. In essence, MALDI MSI is a form of histological screening that does not rely on antibodies or stains2.

<table border="0" cellpadding="0" cellspacing="0" style="width:820px;" width="820">
	<colgroup>
		<col />
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		<col />
	</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

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

If followed correctly, this protocol produces images that clearly represent the gross morphology of the tissue without any scratches or other deformations (Figure 1). The ideal validation for a correctly performed sample preparation, is the ability to distinguish between different physical structures by changing the molecule being viewed (Figure 2).
A good guide for det...

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

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

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

The overall goal of this procedure is to reproducibly prepare a tissue sample for peptide mass spectrometry imaging. This method allows novice mass spectrometry users to develop their own imaging mass spectrometry workflows without going through lengthy empirical determinations. The main advantage of this technique is that it is highly reproducible, simple to implement, and uses cost-effective methodologies.To prepare the nitrocellulose coated slides, pipette 40 microliters of liquid nitrocellulose onto one edge of the conductive indium tin oxide microscope slide. Using a regular glass microscope slide, drag the nitrocellulose under surface tension across the slide to create an even thin-film coating. Allow the nitrocellulose to dry at room temperature for 20 seconds, and then store until needed at room temperature.Next, create custom vapor chambers by cutting a piece of thick blotting paper big enough to fit the bottom half of a standard plastic petri dish. Cut the paper to leave a rectangular strip in the center the same size as a microscope slide. Leave enough excess so that the paper will maintain its position in the petri dish.For FFPE tissue, float mount the sections onto the nitrocellulose pre-coated ITO slides as described in the text protocol. Submerge the slides in fresh xylene to remove residual paraffin for two minutes. Wash the deparaffized samples by submerging the slides in a graded solvent series consisting of a 70%volume for volume ethanol water solution, followed by 100%ethanol, for 30 seconds.Then, submerge the slides into Carnoy's fluid for two minutes. Finally, submerge the slides into a series of 100%ethanol, ultrapure water, and 100%ethanol again, for 30 seconds. Load the sample slides into a plastic slide box that is filled to the top with 20 millimolar tris HCO.Seal the box, and place it in a water bath containing 500 milliliters of water, allowing the box to touch the bottom of the bath. Then heat the box for 15 minutes at 120 degrees celsius in a pressure cooker, which can achieve an operating pressure of 70 kilopascals. Remove the slides, allow them to cool, and let them dry at ambient temperature for 15 minutes.Once hydrolyzed, coat the samples with 10 microliters of trypsin solution in ultrapure water by first pipetting 10 microliters of the solution onto the edge of the tissue section. Then, using the same pipette tip, drag the droplet across the whole surface of the tissue under surface tension. After allowing the samples to dry at ambient temperature, mount them inside the top of the previously constructed vapor chamber with autoclave tape on either edge of the slide.Carefully pipette 600 microliters of a solution containing a one-to-one volume for volume mix of 100%acetonitrile and 50 millimolar ammonium bicarbonate onto the blotting paper finger in the bottom part of the petri dish, until the finger appears to be evenly wet. Pipetting is critically important because incorrect pipetting will result in the delocalization of your matrix and surface analoids which will destroy the spatial information contained within your sample. Place the top half of the vapor chamber on the bottom half, ensuring the paper finger and sample slide align perfectly, and seal the chamber around its equator with paraffin film.Leave the sample over night in a 37 degree celsius incubator to allow complete digestion. Once digested, leave the sample slide on a five-digit microanalytical balance. Mount the slide onto the cooling finger of the sublimation apparatus, and secure it with copper tape in the same way as described for the vapor chamber.Place 300 milligrams of CHCA matrix into a glass petri dish at the bottom of the chamber and spread it evenly to create a thin layer of matrix crystals. Assemble the sublimator. And secure the two halves with the horseshoe clamp.Suspend the assembled unit 15 to 20 centimeters above a sand bath, preheated to 220 degrees celsius, by placing it in a metal ring connected to a retort stand. Connect the chamber to the vacuum source, engage the vacuum, and allow it to stabilize down to approximately 25 millitorr for five minutes. Pack the cooling finger to the top with ice, and add 50 milliliters of water.Allow the apparatus to settle for a further five minutes before proceeding. Lower the chamber onto the surface of the stand, ensuring the sand completely contacts the bottom of the chamber. Leave it for 45 minutes to create an ideal coating of 0.22 milligrams per square centimeter.After 45 minutes, remove the chamber from the sand bath by raising the metal ring, and vent the chamber. Once sublimated, mount the sample slide inside the top of the previously constructed vapor chamber, as before. Carefully pipette 600 microliters of a solution containing a one-to-one volume for volume mix of acetonitrile and trifluoroacetic acid in water onto the blotting paper in the bottom part of the petri dish to ensure an even coating.Next, assemble the chamber, ensuring the paper tab and microscope slide align perfectly. Leave the chamber in a 37 degree celsius incubator for one hour. Scan the slide in a flat-bed scanner to create a digital image.Load the sample into the mass spectrometer, and then analyze using the appropriate software platform. Finally, analyze the samples as described in the text protocol. Shown here is a correctly processed tissue specimen that has been imaged at 50 microns and shows good macro structure and clear difference between white matter and gray matter.Successfully compared samples will show clear differentiation at different tissue locations. Here, there's a clear region of upregulation of the peptide represented by a mass-to-charge ratio of 1, 085 in the white matter region, as represented by white regions. By contrast, this incorrectly prepared sample shows a large-scale level of delocalization.The patterns of the molecules present across the three panels clearly demonstrate that there is no clear definition between biological regions or differences in the abundance of the molecules displayed. After watching this video, you should have a really good idea as to how prepare your own samples for peptide imaging analysis. With proper procedure, this method can be performed within eight hours over two days, allowing for an overnight digestion step.While preparing this methodology, it's important to take care during each step. Any physical damage that happens to the tissue will destroy the spatial information contained within your sample. We had the idea for this methodology when we first started our own IMS space investigations and realized that there were no other definitive methodologies that were published.With small modifications, our methodology can be applied to the analysis of proteins, peptides, lipids, and other small molecules. Following this procedure, other histological techniques such as immunofluorescence and hematoxylin and eosin staining can then be applied to your sample in order to get a reference point for your imaging mass spectrometry data. When working with potentially explosive chemicals, such as liquid nitrocellulose, it's important to maintain the correct safety procedures.Minimizing working volumes and disposing your waste immediately is critically important.

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Research

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Biochemistry

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

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

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

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

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

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

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

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

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

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

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

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

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

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

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

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

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

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

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

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

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

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

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

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...