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).

<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>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>&lt;strong&gt;A. Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1, 4-Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>1.11474</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)&amp;nbsp;</td><td>&amp;nbsp;Carl Roth&amp;nbsp;</td><td>5239.1</td><td>2.5 mM&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (PBS)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>14190169</td><td></td></tr><tr><td>Formic acid (CH&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>AppliChem</td><td>A0748</td><td></td></tr><tr><td>HPLC-grade acetonitrile (ACN, C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;3&lt;/sub&gt;N)</td><td>AppliChem</td><td>A1605</td><td></td></tr><tr><td>HPLC-grade methanol (CH&lt;sub&gt;3&lt;/sub&gt;OH)</td><td>Fisher Scientific</td><td>M/4056/17</td><td></td></tr><tr><td>HPLC-grade water&amp;nbsp;</td><td>AppliChem</td><td>A1589</td><td></td></tr><tr><td>Iodoacetamide (IAA)</td><td>Sigma-Aldrich</td><td>I6125</td><td></td></tr><tr><td>Kalium chloride (KCl)&amp;nbsp;</td><td>&amp;nbsp;Carl Roth&amp;nbsp;</td><td>6781.1</td><td>4.7 mM&amp;nbsp;</td></tr><tr><td>Kalium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>&amp;nbsp;Carl Roth&amp;nbsp;</td><td>3904.2</td><td>1.2 mM&amp;nbsp;</td></tr><tr><td>LC-MS-grade acetic acid</td><td>&amp;nbsp;Carl Roth&amp;nbsp;</td><td>AE69.1</td><td></td></tr><tr><td>Magnesium sulphate (MgSO&lt;sub&gt;4&lt;/sub&gt;)&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;Carl Roth&amp;nbsp;</td><td>261.2</td><td>1.2 mM&amp;nbsp;</td></tr><tr><td>NuPAGE Antioxidant</td><td>Thermo Fisher Scientific (Invitrogen)</td><td>NP0005</td><td></td></tr><tr><td>NuPAGE LDS Sample buffer&amp;nbsp;</td><td>Thermo Fisher Scientific (Invitrogen)</td><td>NP0007</td><td>4x</td></tr><tr><td>NuPAGE MES SDS Running Buffer&amp;nbsp;</td><td>Thermo Fisher Scientific (Invitrogen)</td><td>NP0002</td><td>20x</td></tr><tr><td>NuPAGE Sample reducing agent&amp;nbsp;</td><td>Thermo Fisher Scientific (Invitrogen)</td><td>NP0004</td><td>10x</td></tr><tr><td>SeeBlue Plus2 pre-stained protein standard&amp;nbsp;</td><td>Thermo Fisher Scientific (Invitrogen)</td><td>LC5925</td><td></td></tr><tr><td>Sequencing grade modified trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>&amp;nbsp;Carl Roth&amp;nbsp;</td><td>9265.2</td><td>118.3 mM&amp;nbsp;</td></tr><tr><td>Sodium hydrogen carbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>&amp;nbsp;Carl Roth&amp;nbsp;</td><td>965.3</td><td>25 mM&amp;nbsp;</td></tr><tr><td>Trifluoroacetic acid (TFA,&amp;nbsp; C&lt;sub&gt;2&lt;/sub&gt;HF&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Merck Millipore</td><td>108178</td><td></td></tr><tr><td>&amp;alpha;-(D)-(+)- Glucose monohydrate</td><td>&amp;nbsp;Carl Roth&amp;nbsp;</td><td>6780.1</td><td>11 mM&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;B. Reagents and Kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.5 mm zirconium oxide beads&amp;nbsp;</td><td>Next Advance</td><td>ZROB05</td><td></td></tr><tr><td>1.0 mm zirconium oxide beads&amp;nbsp;</td><td>Next Advance</td><td>ZROB10</td><td></td></tr><tr><td>Colloidal Blue Staining&amp;nbsp; Kit</td><td>Thermo Fisher Scientific (Invitrogen)</td><td>LC6025</td><td>To stain 25 mini gels per kit</td></tr><tr><td>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><td>Pierce Bicinchoninic Acid (BCA) Protein Assay Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>ProteoExtract Transmembrane Protein Extraction Kit, TM-PEK</td><td>Merck Millipore</td><td>71772-3</td><td>20 reactions per kit</td></tr><tr><td>Tissue Protein Extraction Reagent (T-PER)</td><td>Thermo Scientific</td><td>78510</td><td></td></tr><tr><td>&lt;strong&gt;C. Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well V-bottom plates</td><td>Greiner Bio-One</td><td>651180</td><td></td></tr><tr><td>Corning 96-well flat-bottom plates</td><td>Sigma-Aldrich</td><td>CLS3595-50EA</td><td></td></tr><tr><td>Disposable microtome blades</td><td>pfm Medical</td><td>207500014</td><td></td></tr><tr><td>Disposable scalpels #21</td><td>pfm Medical</td><td>200130021</td><td></td></tr><tr><td>Dissection pins&amp;nbsp;</td><td>Carl Roth</td><td>PK47.1</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>&amp;nbsp;Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Falcon conical centrifuge tubes (50 mL)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Mayo scissors, Tough cut</td><td>&amp;nbsp;Fine Science Tools</td><td>14130-17</td><td></td></tr><tr><td>Precision tweezers</td><td>&amp;nbsp;Fine Science Tools</td><td>11251-10</td><td>Type 5</td></tr><tr><td>Precision tweezers, straight with extra fine tips</td><td>Carl Roth</td><td>LH53.1</td><td>Type 5</td></tr><tr><td>Self-adhesive sealing films for microplates</td><td>Ratiolab (vWR)</td><td>RATI6018412</td><td></td></tr><tr><td>Standard pattern forceps</td><td>&amp;nbsp;Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Student Vannas spring scissors</td><td>&amp;nbsp;Fine Science Tools</td><td>91501-09</td><td></td></tr><tr><td>Vannas capsulotomy scissors&amp;nbsp;&amp;nbsp;</td><td>Geuder</td><td>19760</td><td>&amp;nbsp;Straight, 77 mm</td></tr><tr><td>ZipTip&lt;sub&gt;C18&lt;/sub&gt; pipette tips</td><td>Merck Millipore</td><td>ZTC18S096</td><td></td></tr><tr><td>&lt;strong&gt;D. Equipment and devices&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>150 &amp;times; 0.5 mm BioBasic C18 column</td><td>Thermo Scientific, Rockford, USA</td><td>72105-150565</td><td></td></tr><tr><td>30 &amp;times; 0.5 mm BioBasic C18 pre-column&amp;nbsp;</td><td>Thermo Scientific, Rockford, USA</td><td>72105-030515</td><td></td></tr><tr><td>Amicon Ultra-0.5 3K Centrifugal Filter Devices&amp;nbsp;</td><td>Merck Millipore</td><td>UFC500396</td><td>Pack of 96.</td></tr><tr><td>Analytical balance</td><td>Sartorius</td><td>H51</td><td></td></tr><tr><td>Autosampler&amp;nbsp;</td><td>CTC Analytics AG, Zwingen, Switzerland</td><td>HTS Pal</td><td></td></tr><tr><td>BBY24M Bullet Blender Storm&amp;nbsp;</td><td>Next Advance</td><td>NA-BB-25</td><td></td></tr><tr><td>Eppendorf concentrator, model 5301</td><td>Sigma-Aldrich</td><td>Z368172</td><td></td></tr><tr><td>Eppendorf microcentrifuge, model 5424</td><td>Fisher Scientific</td><td>05-403-93</td><td>Non-refrigerated</td></tr><tr><td>Heraeus Primo R Centrifuge</td><td>Thermo Scientific</td><td>75005440</td><td>Refrigerated</td></tr><tr><td>Labsonic M Ultrasonic homogenizer&amp;nbsp;</td><td>Sartorius</td><td>BBI-8535027</td><td></td></tr><tr><td>LC-MS pump, model Rheos Allegro</td><td>Thermo Scientific, Rockford, USA</td><td>22080</td><td></td></tr><tr><td>LTQ Orbitrap XL mass spectrometer&amp;nbsp;</td><td>Thermo Scientific, Bremen, Germany</td><td></td><td></td></tr><tr><td>Multiskan Ascent plate reader&amp;nbsp;</td><td>Thermo Labsystems</td><td>v2.6</td><td></td></tr><tr><td>Rotator with vortex&amp;nbsp;</td><td>neoLab</td><td>7-0045</td><td></td></tr><tr><td>Titanium probe (&amp;Oslash; 0.5 mm, 80 mm long)</td><td>Sartorius</td><td>BBI-8535612</td><td></td></tr><tr><td>Ultrasonic bath, type RK 31</td><td>Bandelin</td><td>329</td><td></td></tr><tr><td>Xcell Surelock Mini Cell</td><td>Life Technologies</td><td>El0001</td><td></td></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 ...

sample-preparation-for-mass-spectrometry-based-proteomics-analysis

Research

JoVE Journal

Biology

11.2K Views.  University Medical Centre of the Johannes Gutenberg University Mainz. De voorbereiding van hoogwaardige massaspectrometrie-compatibele monsters voor uitgebreide proteomeanalyse van oculaire monsters is van cruciaal belang om de moleculaire mechanismen en signaleringstrajecten die betrokken zijn bij gezondheid en ziekte te verduidelijken. De beschreven werkstroom vertegenwoordigt een eenvoudige maar robuuste benadering van strenge monsterbereidingsstappen die specifiek worden verzorgd voor de analyse van massabesprekte monsters zoals oculaire microbloedvaten. Ho...

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

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Medicine

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Biochemistry

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

Cancer Research

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

Chemistry

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

Neuroscience

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson&#39;s disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.

A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the research laboratory. Here, we developed a protocol for acquiring high-quality liquid biopsies for molecular analyses from the aqueous humor and the vitreous from patients undergoing eye surgery. In this workflow, a Mobile Operating Room Lab Interface (MORLI) cart equipped with a computer, a barcode scanner, and lab instruments, including onboard cold storage, is used to obtain and archive human biological samples. A web-based data privacy-compliant database enables annotating each sample over its lifetime, and a cartesian coordinate system allows tracking each barcoded specimen in storage, enabling quick and accurate retrieval of samples for downstream analyses. Molecular characterization of human tissue samples not only serves as a diagnostic tool (e.g., to distinguish between infectious endophthalmitis and other non-infectious intraocular inflammation) but also represents an important component of translational research, allowing the identification of new drug targets, development of new diagnostic tools, and personalized therapeutics.

This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the ...

Non-Invasive Biomarker Discovery for Tear Proteomics Using LC-MS

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

Author Spotlight: Advancing Tear Fluid Analysis Using a Standardized Protocol for Proteomics Research 

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...

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

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