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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			<td height="21" style="height:21px;">Heraeus Primo R Centrifuge</td>
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			<td height="21" style="height:21px;">LC-MS pump, model Rheos Allegro</td>
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			<td height="21" style="height:21px;">LTQ Orbitrap XL mass spectrometer&#160;</td>
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			<td style="width:120px;">v2.6</td>
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			<td height="21" style="height:21px;">Rotator with vortex&#160;</td>
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			<td height="21" style="height:21px;">Titanium probe (&#216; 0.5mm, 80mm long)</td>
			<td style="width:281px;">Sartorius</td>
			<td style="width:120px;">BBI-8535612</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonic bath, type RK 31</td>
			<td style="width:281px;">Bandelin</td>
			<td style="width:120px;">329</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Xcell Surelock Mini Cell</td>
			<td>Life Technologies</td>
			<td>El0001</td>
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		</tr>
	</tbody>
</table>

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 ...

Research

JoVE Journal

Biology

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

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Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...