Name: sample preparation for mass-spectrometry-based proteomics analysis of ocular microvessels
Uploaded: 2019-02-22T14:30:00
Description: Watch this Scientific Journal Video about Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels at JoVE.com

Dr. Manicam is supported by the Internal University Research Funding (Stufe 1) from the University Medical Centre of the Johannes Gutenberg University Mainz and a grant from the Deutsche Forschungsgemeinschaft (MA 8006/1-1).

All experimental procedures using animal samples were performed in strict adherence to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and by institutional guidelines. This study was conducted and approved at the Department of Ophthalmology, University Medical Centre Mainz.
NOTE: Porcine eyes together with optic nerve and extraocular tissues were obtained fresh from the local abattoir immediatel...

<ol>
	<li>Mandal, N., Heegaard, S., Prause, J. U., Honor&#233;, B., Vorum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+proteomics+with+emphasis+on+two-dimensional+gel+electrophoresis+and+mass+spectrometry.">Ocular proteomics with emphasis on two-dimensional gel electrophoresis and mass spectrometry.</a> Biological Procedures Online. 12, 56-88 (2010).</li><li>Gregorich, Z. R., Ge, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+proteomics+in+health+and+disease:+Challenges+and+opportunities.">Top-down proteomics in health and disease: Challenges and opportunities.</a> Proteomics. 14, 1195-1210 (2014).</li><li>Aebersold, R., Mann, 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+spectrometry-based+proteomics.">Mass spectrometry-based proteomics.</a> Nature. 422, 198-207 (2003).</li><li>Aebersold, R., Mann, 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+exploration+of+proteome+structure+and+function.">Mass-spectrometric exploration of proteome structure and function.</a> Nature. 537, 347-355 (2016).</li><li>Cehofski, L. J., Mandal, N., Honor&#233;, B., Vorum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+platforms+in+vitreoretinal+proteomics.">Analytical platforms in vitreoretinal proteomics.</a> Bioanalysis. 6, 3051-3066 (2014).</li><li>Manicam, C., 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=Proteomics+Unravels+the+Regulatory+Mechanisms+in+Human+Tears+Following+Acute+Renouncement+of+Contact+Lens+Use:+A+Comparison+between+Hard+and+Soft+Lenses.">Proteomics Unravels the Regulatory Mechanisms in Human Tears Following Acute Renouncement of Contact Lens Use: A Comparison between Hard and Soft Lenses.</a> Scientific Reports. 8, 11526 (2018).</li><li>Perumal, N., Funke, S., Pfeiffer, N., Grus, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+lacrimal+proline-rich+protein+4+(PRR+4)+in+human+tear+proteome.">Characterization of lacrimal proline-rich protein 4 (PRR 4) in human tear proteome.</a> Proteomics. 14, 1698-1709 (2014).</li><li>Perumal, N., Funke, S., Pfeiffer, N., Grus, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+analysis+of+human+tears+from+aqueous-deficient+and+evaporative+dry+eye+patients.">Proteomics analysis of human tears from aqueous-deficient and evaporative dry eye patients.</a> Scientific Reports. 6, 29629 (2016).</li><li>Perumal, N., Funke, S., Wolters, D., Pfeiffer, N., Grus, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+reflex+tear+proteome+reveals+high+expression+of+lacrimal+proline-rich+protein+4+(PRR4).">Characterization of human reflex tear proteome reveals high expression of lacrimal proline-rich protein 4 (PRR4).</a> Proteomics. 15, 3370-3381 (2015).</li><li>Perumal, 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=Characterization+of+the+human+aqueous+humour+proteome:+A+comparison+of+the+genders.">Characterization of the human aqueous humour proteome: A comparison of the genders.</a> PloS ONE. 12, 0172481 (2017).</li><li>Hayreh, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+ciliary+artery+circulation+in+health+and+disease+the+Weisenfeld+lecture.">Posterior ciliary artery circulation in health and disease the Weisenfeld lecture.</a> Investigative Ophthalmology &amp; Visual Science. 45, 749-757 (2004).</li><li>Zeitz, O., 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=Glaucoma+progression+is+associated+with+decreased+blood+flow+velocities+in+the+short+posterior+ciliary+artery.">Glaucoma progression is associated with decreased blood flow velocities in the short posterior ciliary artery.</a> British Journal of Ophthalmology. 90, 1245-1248 (2006).</li><li>Verma, N., Rettenmeier, A. W., Schmitz-Spanke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+use+of+Sus+scrofa+(pig)+as+a+model+system+for+proteomic+studies.">Recent advances in the use of Sus scrofa (pig) as a model system for proteomic studies.</a> Proteomics. 11, 776-793 (2011).</li><li>Foulds, W. S., Kek, W. K., Luu, C. D., Song, I. C., Kaur, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+of+selective+retinal+capillary+closure+induced+by+embolization+with+fluorescent+microspheres.">A porcine model of selective retinal capillary closure induced by embolization with fluorescent microspheres.</a> Investigative Ophthalmology &amp; Visual Science. 51, 6700-6709 (2010).</li><li>Sanchez, I., Martin, R., Ussa, F., Fernandez-Bueno, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parameters+of+the+porcine+eyeball.">The parameters of the porcine eyeball.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 249, 475-482 (2011).</li><li>Manicam, C., Perumal, N., Pfeiffer, N., Grus, F. H., Gericke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+insight+into+the+proteome+landscape+of+the+porcine+short+posterior+ciliary+arteries:+Key+signalling+pathways+maintaining+physiologic+functions.">First insight into the proteome landscape of the porcine short posterior ciliary arteries: Key signalling pathways maintaining physiologic functions.</a> Scientific Reports. 6, 38298 (2016).</li><li>Shevchenko, A., Tomas, H., Havli, J., Olsen, J. V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+digestion+for+mass+spectrometric+characterization+of+proteins+and+proteomes.">In-gel digestion for mass spectrometric characterization of proteins and proteomes.</a> Nature Protocols. 1, 2856-2860 (2006).</li><li>Feist, P., 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=Proteomic+challenges:+sample+preparation+techniques+for+microgram-quantity+protein+analysis+from+biological+samples.">Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples.</a> International Journal of Molecular Sciences. 16, 3537-3563 (2015).</li><li>Cox, B., Emili, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+subcellular+fractionation+and+protein+extraction+for+use+in+mass-spectrometry-based+proteomics.">Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics.</a> Nature Protocols. 1, 1872-1878 (2006).</li><li>Zhang, L., 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=Proteomic+analysis+of+mouse+liver+plasma+membrane:+use+of+differential+extraction+to+enrich+hydrophobic+membrane+proteins.">Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins.</a> Proteomics. 5, 4510-4524 (2005).</li><li>Zhou, 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=Improved+recovery+and+identification+of+membrane+proteins+from+rat+hepatic+cells+using+a+centrifugal+proteomic+reactor.">Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor.</a> Molecular &amp; Cellular Proteomics. 10, 111 (2011).</li><li>de la Cuesta, F., Mourino-Alvarez, L., Baldan-Martin, M., Moreno-Luna, R., Barderas, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+proteomics+to+the+management+of+vascular+disorders.">Contribution of proteomics to the management of vascular disorders.</a> Translational Proteomics. 7, 3-14 (2015).</li><li>Cottingham, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1DE+proves+its+worth...+again.">1DE proves its worth... again.</a> Journal of Proteome Research. 9, 1636 (2010).</li></ol>

Comprehensive proteome profiling of a diverse range of ocular samples is an important and indispensable first step to elucidate the molecular mechanisms and signaling pathways implicated in health and disease. In order to obtain high quality data and to ensure the reproducibility of results obtained from these analyses, the preceding sample preparation steps are crucial, as highlighted in a review by Mandal et al. that discussed in-depth the sample processing procedures for different parts of the eye employing two-dimens...

The advancement in the field of proteomics, which permits integrated and unsurpassed data collection power, has greatly revolutionized our understanding of the molecular mechanisms underlying certain disease conditions as well as in reflecting the physiological state of a specific cell population or tissue1,2,3,4. Proteomics has also proved to be an important platform in ophthalmic research owing to the sensitivity and unbiased analysis of different ocular samples that facilitated identification of potential disease markers for eventual diagnosis and prognosis, as evidenced elegantly by many studies in recent years, including some of ours1,5,6,7,8,9,10. However, it is often difficult to obtain human samples for proteomic analyses due to ethical reasons, especially considering the need for control material from healthy individuals for reliable comparative analyses. On the other hand, it is also challenging to obtain sufficient amount of samples for optimal and reliable mass spectrometric analyses. This is particularly crucial for mass-limited biological materials such as the micro-blood vessels of the eye. One such major retrobulbar blood vessel that plays pivotal roles in the regulation of ocular blood flow is the short posterior ciliary artery (sPCA). Any perturbation or anomalies in this vascular bed may result in severe clinical repercussions, which can lead to the pathogenesis of several sight-threatening diseases such as glaucoma and nonarteritic anterior ischemic optic neuropathy (NAION)11,12. However, there is a lack of studies elucidating the proteome changes in this arterial bed due to the above-mentioned drawbacks. Therefore, in recent years, the house swine (Sus scrofa domestica Linnaeus, 1758) has emerged as a good animal model in ophthalmic research owing to the high morphologic and phylogenetic similarities between humans and pigs13,14,15. Porcine ocular samples are easily available and most importantly, are more accurate representation of human tissues.
Considering the important role of these blood vessels in the eye, as well as the dearth of methodology catered for efficient protein extraction and analyses from these microvessels, we have previously characterized the proteome of the porcine sPCA using an in-house protocol that resulted in the identification of a high number of proteins16. Based on this study, we have further optimized and described in-depth our methodology in this article, which allows proteome analysis from minute amounts of samples using the porcine sPCA as model tissue. Albeit the main aim of this study was to establish a MS-compatible methodology for mass-limited ocular blood vessels, we have provided substantial experimental evidence that the described workflow can also be broadly applied to various tissue-based samples.
It is envisioned that this workflow will be instrumental for preparation of high-quality MS-compatible samples from small quantities of materials for comprehensive proteome analyses.

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			<td height="21" style="height:21px;width:436px;">A. Chemicals</td>
			<td style="width:281px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1, 4-Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:120px;">1.11474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium bicarbonate (ABC, CH&#8325;NO&#8323;)</td>
			<td>Sigma-Aldrich</td>
			<td>5.33005</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Calcium chloride dihydrate (CaCl2)&#160;</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>5239.1</td>
			<td>2.5 mM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s phosphate-buffered saline (PBS)&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Formic acid (CH2O2)</td>
			<td style="width:281px;">AppliChem</td>
			<td style="width:120px;">A0748</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HPLC-grade acetonitrile (ACN, C2H3N)</td>
			<td style="width:281px;">AppliChem</td>
			<td style="width:120px;">A1605</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HPLC-grade methanol (CH3OH)</td>
			<td style="width:281px;">Fisher Scientific</td>
			<td style="width:120px;">M/4056/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC-grade water&#160;</td>
			<td style="width:281px;">AppliChem</td>
			<td style="width:120px;">A1589</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodoacetamide (IAA)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:120px;">I6125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kalium chloride (KCl)&#160;</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>6781.1</td>
			<td>4.7 mM&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Kalium dihydrogen phosphate (KH2PO4)</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>3904.2</td>
			<td>1.2 mM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LC-MS-grade acetic acid</td>
			<td style="width:281px;">&#160;Carl Roth&#160;</td>
			<td style="width:120px;">AE69.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium sulphate (MgSO4)&#160;&#160;</td>
			<td>&#160;Carl Roth&#160;</td>
			<td style="width:120px;">261.2</td>
			<td>1.2 mM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE Antioxidant</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td style="width:120px;">NP0005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE LDS Sample buffer&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td>NP0007</td>
			<td>4x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE MES SDS Running Buffer&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td>NP0002</td>
			<td>20x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE Sample reducing agent&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td>NP0004</td>
			<td>10x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SeeBlue Plus2 pre-stained protein standard&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td style="width:120px;">LC5925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sequencing grade modified trypsin</td>
			<td style="width:281px;">Promega</td>
			<td style="width:120px;">V5111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>9265.2</td>
			<td>118.3 mM&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium hydrogen carbonate (NaHCO3)</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>965.3</td>
			<td>25 mM&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Trifluoroacetic acid (TFA,&#160; C2HF3O2)</td>
			<td style="width:281px;">Merck Millipore</td>
			<td style="width:120px;">108178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-(D)-(+)- Glucose monohydrate</td>
			<td>&#160;Carl Roth&#160;</td>
			<td>6780.1</td>
			<td>11 mM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B. Reagents and Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5mm zirconium oxide beads&#160;</td>
			<td style="width:281px;">Next Advance</td>
			<td style="width:120px;">ZROB05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0mm zirconium oxide beads&#160;</td>
			<td style="width:281px;">Next Advance</td>
			<td style="width:120px;">ZROB10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colloidal Blue Staining&#160; Kit</td>
			<td style="width:281px;">Thermo Fisher Scientific (Invitrogen)</td>
			<td style="width:120px;">LC6025</td>
			<td>To stain 25 mini gels per kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE 4-12 % Bis-Tri gels</td>
			<td>Thermo Fisher Scientific (Invitrogen)</td>
			<td>NP0321BOX</td>
			<td>1.0 mm, 10-well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Bicinchoninic Acid (BCA) Protein Assay Kit&#160;</td>
			<td style="width:281px;">Thermo Fisher Scientific</td>
			<td style="width:120px;">23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProteoExtract Transmembrane Protein Extraction Kit, TM-PEK</td>
			<td style="width:281px;">Merck Millipore</td>
			<td style="width:120px;">71772-3</td>
			<td>20 reactions per kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Protein Extraction Reagent (T-PER)</td>
			<td style="width:281px;">Thermo Scientific</td>
			<td style="width:120px;">78510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C. Tools</td>
			<td style="width:281px;"></td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well V-bottom plates</td>
			<td style="width:281px;">Greiner Bio-One</td>
			<td style="width:120px;">651180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 96-well flat-bottom plates</td>
			<td style="width:281px;">Sigma-Aldrich</td>
			<td style="width:120px;">CLS3595-50EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable microtome blades</td>
			<td>pfm Medical</td>
			<td>207500014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable scalpels #21</td>
			<td>pfm Medical</td>
			<td>200130021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection pins&#160;</td>
			<td style="width:281px;">Carl Roth</td>
			<td style="width:120px;">PK47.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extra Fine Bonn Scissors</td>
			<td style="width:281px;">&#160;Fine Science Tools</td>
			<td style="width:120px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon conical centrifuge tubes (50 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayo scissors, Tough cut</td>
			<td style="width:281px;">&#160;Fine Science Tools</td>
			<td style="width:120px;">14130-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision tweezers</td>
			<td style="width:281px;">&#160;Fine Science Tools</td>
			<td>11251-10</td>
			<td>Type 5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision tweezers, straight with extra fine tips</td>
			<td style="width:281px;">Carl Roth</td>
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sample preparation for mass-spectrometry-based proteomics analysis of ocular microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Limited sample availability is one of the major drawbacks in ophthalmic research. Correspondingly, extraction methods for optimum protein yield from small amounts of samples such as ocular blood vessels are often debatable. To date, there is a paucity of methods catered particularly for protein extraction from retrobulbar blood vessels. Therefore, as a first step in method optimization and as a proof-of-principle to compare the efficacy and robustness of several commonly employed protein ...

Watch this Scientific Journal Video about Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels at JoVE.com

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

The preparation of high-quality mass-spectrometry-compatible samples for comprehensive proteome analysis of ocular samples is critical to elucidate the molecular mechanisms and signaling pathways implicated in health and disease. The described work flow represents a simple yet robust approach to stringent sample preparation steps catered specifically for analysis of mass-limited samples such as ocular micro blood vessels. Although the main aim of the study is to establish an MS-compatible methodology for ocular blood vessels, the described work flow can be broadly applied to various cell-and tissue-based samples.Generally, individuals new to this method may struggle because the identification and isolation of the chosen short posterior ciliary artery, or SPCA branches, can be challenging. Fresh porcine eyes, together with optic nerve and extraocular tissues, were obtained from the local abattoir immediately post-mortem. Place the eye globe in a dissection chamber containing ice-cold Krebs-Henseleit buffer.Carefully cut away surrounding muscle and tissues with a pair of sharp Mayo scissors. Make an incision with a scalpel, and cut the globe along the equatorial plane with a pair of sharp Mayo scissors until the eye is separated into the anterior and posterior halves. Remove as much vitreous body as possible from the posterior half of the eye using a pair of standard pattern forceps.After using dissection pins to carefully pin down the posterior half of the eye, gently cut away the connective tissues surrounding the optic nerve, to expose the underlying retrobulbar vasculature with a pair of student Vannas spring scissors. Isolate the paraoptic and distal short posterior ciliary artery together with the surrounding connective tissues with a pair of type five precision tweezers and Vannas capsulotomy scissors. Gently remove connective tissues from the arterial segments using extra fine tipped tweezers and scissors before rinsing the isolated arteries in ice-cold PBS to remove contaminants and blood residues.Pool arteries from two eyes to obtain one biological replicate, then weigh the samples using an analytical balance. To each tube, add a mixture of 0.5 and one-millimeter zirconium oxide beads followed by tissue protein extraction reagent. Now, load the sample tubes into a blender homogenizer.Set the timer to two minutes, set the speed level to six, and start homogenization. After running, check the samples for complete homogenization and keep samples on ice in between each run. Repeat the cycle until samples are completely homogenized.Carefully pipette homogenate into fresh microcentrifuge tubes. Centrifuge the samples at 10, 000 times G for 20 minutes at four degrees Celsius to pellet insoluble proteins. Carefully pipette the supernatant containing the soluble proteins without touching the pellet layer, and transfer into fresh microcentrifuge tubes.Add 200 microliters of Extraction Buffer 2A and five microliters of protease inhibitor cocktail to each pellet sample. Then, suspend the pellet several times with a pipette. Use an ultrasonic homogenizer to completely homogenize the pellet.Set the amplitude to 60 and cycle to one. Use a probe made of titanium, which is appropriate for homogenization of samples with small volumes. Immerse the probe in the pellet and extraction buffer mixture on ice, and press the start button.Sonicate the sample until the pellet clumps are completely homogenized, pausing for a few seconds between each sonication. Check for complete homogenization visually. Mix the homogenate several times with a pipette to ensure that there are no clumps.Use centrifugal filter devices with three-kilodalton cutoff for this procedure. Insert the three-kilodalton cutoff filter unit into a microcentrifuge tube. Pipette 200 microliters of sample homogenate to one filter device, and add 200 microliters of deionized water into the same filter.After capping it securely, place the filter unit into a centrifuge, and spin for 14, 000 times G for 15 minutes at four degrees Celsius. After centrifugation, separate the filter unit containing the sample concentrate from the microcentrifuge tube containing the filtrate, discarding the filtrate. Reconstitute the concentrate by adding 400 microliters of deionized water into the filter unit.After repeating filtration three times, carefully pipette the cleaned sample concentrate into a clean microtube. Prepare the samples for one-dimensional gel electrophoresis as listed in the text protocol, and mix well with a pipette. Heat the samples at 70 degrees Celsius in a dry block heater for 15 minutes, and cool to room temperature.Carefully load 50 micrograms of sample per lane using a pipette, as well as the prestained protein standard as a molecular mass marker. Run the gels for approximately 60 minutes at a constant voltage of 175 volts. At the end of the run, carefully remove the gel from the cassette plate using a gel knife, and transfer the gel into a gel staining box.Perform fixing and staining of the gel as described in the text protocol. Shake the gels in the staining solution overnight for best overall results. Carefully decant the staining solution and replace with 200 milliliters of deionized water before shaking the gels for at least seven to eight hours in water to clear the background.Excise the protein bands from the gel with clean new microtome blades. Cut the band into small pieces, and carefully transfer the gel pieces into 1.5-milliliter microcentrifuge tubes. Add 500 microliters of destaining solution containing 100-millimolar ammonium bicarbonate and acetonitrile.Incubate the samples at room temperature for 30 minutes, with occasional shaking. Carefully pipette out the destaining solution. Check visually for any tubes with residual stain, and repeat this step if gel pieces are still stained blue.Add approximately 400 microliters of freshly prepared DTT solution, and incubate at 56 degrees Celsius for 30 minutes. After discarding the reducing solution with a pipette, add approximately 400 microliters of freshly prepared IAA solution, and incubate in the dark at room temperature for 30 minutes. Remove the alkylating buffer with a pipette and discard.Add 500 microliters of neat acetonitrile at room temperature for 10 to 15 minutes, until the gel pieces shrink and become opaque. Pipette out the acetonitrile, and air-dry the gel pieces for five to 10 minutes under the hood. Then, pipette 50 microliters of trypsin solution into each tube to completely cover the gel pieces, and incubate the tubes at four degrees Celsius.After 30 minutes, add sufficient volume of trypsin buffer to completely cover the gel pieces if necessary. Incubate the samples overnight at 37 degrees Celsius. To perform peptide extraction, carefully pipette the extracted peptide solution from the tubes and transfer to clean microtubes.Dry the supernatant in a centrifugal vacuum evaporator. Add 100 microliters of extraction buffer to each tube with gel pieces, and incubate for 30 minutes with shaking. Pipette the supernatant into the same microtubes containing the extracted peptides according to their respective bands, and dry down in a vacuum centrifuge.Proceed to peptide purification and liquid chromatography-electrospray ionization MS/MS analyses as described in the text protocol. The total amount of proteins in samples extracted with each type of detergent is depicted here. The highest yield came from tissues extracted with tissue protein extraction buffer, followed by DDM, CHAPS, ASB-14, and the lowest yield from ACN and TFA.Consistently, the total proteins identified were also the highest in the tissue protein extraction buffer extracted sample, and followed same trend as the total yield. Shown here is the comparison of the one-dimension gel electrophoresis protein profiles of SPCA, before and after being subjected to the optimized sample preparation and cleaning steps. Overall, a high degree of smearing and poor separation of the protein bands was observed at lane three.This profile demonstrates that the samples may contain extraction reagent, and also contaminants such as lipids and cellular debris. However, the SPCA samples that were separated into supernatant and pellet, and then subjected to the optimized protocol, resulted in exemplary one-dimensional gel electrophoresis profiles. The optimized method for rapid, robust, and efficient protein extraction from ocular microvessels can also be readily applied to other tissue-based samples.Shown here are protein profiles of the supernatant and the pellet of murine brain and cardiac tissue samples. While attempting this procedure, it is important to remember to subject the samples to complete homogenization, to separate the supernatant from the pellet, and to remove the contaminants and the extraction reagents. After its development, this technique paved the way for researchers in the field of experimental and translational ophthalmology to thoroughly explore the proteome of oscular vasculature using porcine eyes as a model.

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