We thank R. Bartenschlager (University of Heidelberg) for the Jc1 constructs, C.M. Rice (Rockefeller University) for the Huh7.5 cells, J. McLauchlan (Medical Research Council Virology Unit) for the JFH1 construct, T. Wakita (National Institute of Infectious Diseases, Japan) for the JFH1, and B. Webster and W.C. Greene (Gladstone Institute of Virology and Immunology) for the HCVcc reporter constructs. This work was supported by funds from the DFG (HE 6889/2-1 (EH), INST 337/15-1 2013, and INST 337/16-1 2013 (HS)). The Heinrich Pette Institute, Leibniz Institute for Experimental Virology is supported by the Free and Hanseatic City of Hamburg and the Federal Ministry of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Preparation of Media for Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)
NOTE: Here, the SILAC Protein Quantitation Kit - DMEM supplemented with 50 mg of 13C6 L-Arginine-HCl was used for SILAC labeling. The dialyzed Fetal Calf Serum (FCS) is provided with the SILAC Protein Quantitation Kit.
<ol>
	<li>Remove 50 mL from each bottle of DMEM medium and add 50 mL of dialyzed FCS.</li>
	<li>Dissolve 50 mg of 13C6 L-Lysine-2HCl and 50 mg of 13C6 L-Arginine-HCl in 1 mL of medium. Mix thoroughly and add the amino acids to the DMEM + FCS medium.</li>
	<li>Add 1x Pen/Strep and 1x L-glutamine substitute. Sterile-filter the medium using a 0.45 &#181;m filter. Label the bottle as &#34;heavy&#34; SILAC medium.</li>
	<li>To prepare the &#34;light&#34; medium, repeat steps 1.1 - 1.3 using 50 mg of L-Arginine-HCl and 50 mg of L-Lysine-2HCl. Label the bottle as &#34;light&#34; SILAC medium.</li>
</ol>
2. SILAC-labeling and Amino Acid Incorporation Control
<ol>
	<li>Trypsinize cells (1x Trypsin-EDTA) and split 1 x 105 Huh7.5 cells into 2 wells of a 6-well culture plate containing 2 mL of medium, one well with the &#34;heavy&#34; SILAC medium and one with the &#34;light&#34; SILAC medium.</li>
	<li>Culture the cells for at least 6 passages (split ratio: 1:6); after 6 passages, the incorporation of the &#34;heavy&#34; amino acids should be more than 95%.</li>
	<li>Harvest 1 x 106 cells of the &#34;heavy&#34;- and &#34;light&#34;-labeled cell population to analyze the incorporation efficacy. Wash the cells in 1x PBS and pellet the cells by centrifugation for 5 min at 160 x g and 4 &#176;C.</li>
	<li>Resuspend the cell pellets in 150 &#181;L of MS-buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, and 1 mM EDTA supplemented with 1x protease inhibitor cocktail) and incubate the cells on ice for 30 min. Lyse the cells by sonication at 4 &#176;C. 
	NOTE: The following are the sonication settings used here: timer, hold; output control, 8; duty cycle, 80%; and 2 x 30 pulses.</li>
	<li>Remove the cell debris by centrifugation for 10 min at 11,000 x g and 4 &#176;C. Transfer the supernatant to a new tube and determine the protein concentration with a detergent-compatible protein assay.</li>
	<li>Mix 75 &#181;g of protein with 6x sample buffer (375 mM Tris-HCl, pH 6.8, 25.8% glycerol, 123 mg/mL SDS, 600 &#181;g/mL bromophenol blue, and 60 &#181;L/mL &#946;-mercaptoethanol), boil at 95 &#176;C for 5 min, and separate the proteins by SDS-PAGE in SDS running buffer (3.02 g/L Tris base, 18.8 g/L glycine, and 1 g/L SDS) at 180 V for approximately 1 h or according to the manufacturers&#39; instructions.</li>
	<li>Transfer the gel into colloidal Coomassie staining solution. Excise the same protein band from each lane. Digest the proteins with trypsin and analyze the incorporation efficacy by MS analysis, as described22.</li>
</ol>
3. Lipid Droplet Isolation of SILAC-labeled Huh7.5 Cells
<ol>
	<li>Infect one population of cells (i.e.&#160;those labeled with &#34;light&#34; amino acids) with an HCV reporter virus by incubating with virus stocks (e.g., Jc1NS5AB-mKO2-BSD, MOI 1) for 4 h at 37 &#176;C, as described21. 
	NOTE: Jc1NS5AB-mKO2-BSD is an HCV virus carrying a fluorescence reporter (monomeric Kusabira Orange 2, mKO2) to monitor infection rates followed by a Blasticidin Resistance Gene (BSD) between a duplicated NS5A-NS5B cleavage site, described previously21,23. Work with HCV requires BSL2+ (USA) or S3** (Germany) biosafety-level containment and practices. Instead of infection with HCV, cells can be infected with different pathogens.</li>
	<li>Expand HCV-infected &#34;light&#34; cells and noninfected &#34;heavy&#34; cells. During the passaging of the cultures, fix an aliquot with 4% paraformaldehyde (PFA) in PBS and determine the HCV infection rates by flow cytometry of the fluorescent marker protein (e.g., mKO2), as described21; the infection rates should be higher than 90% for lipid droplet isolation. NOTE:&#160;If using the Jc1NS5AB-mKO2-BSD HCV strain, add 10 &#181;g/mL blasticidin S to the &#34;light&#34; medium to select for HCV-positive cells.</li>
	<li>1 d prior to lipid droplet isolation, wash the cells with PBS, trypsinize, resuspend in &#34;light&#34; and &#34;heavy&#34; medium, and count the cells using a Neubauer counting chamber. Seed 7 x 106 cells of each population in a 150 cm2 cell culture dish. Prepare at least 5 dishes per &#34;light&#34; and &#34;heavy&#34; cell population. Culture the cells in 30 mL of medium/dish O/N.</li>
	<li>Remove the medium and wash the cells in 1x PBS. Detach the cells in 1x PBS using a cell scraper.</li>
	<li>Count both cell populations using a Neubauer counting chamber and pool equal cell numbers in a 50 mL centrifuge tube. Pellet the cells by centrifugation for 5 min at 160 x g and 4 &#176;C.</li>
	<li>Remove the PBS and resuspend the cell pellet in 1 mL of sucrose buffer (0.25 M sucrose, 1 mM EDTA, and 1 mM DTT, supplemented with protease inhibitor cocktail). Transfer the cell suspension to a tight-fitting Dounce homogenizer and lyse the cells with 200 strokes on ice. 
	NOTE: Confirm complete cell lysis using trypan blue staining.</li>
	<li>Transfer the lysate into a 1.5 mL microfuge tube and spin down the nuclei and cell debris for 10 min at 1,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, store an aliquot of 25 &#181;L of the post-nuclear fraction (PNS) at -20 &#176;C as an input control.</li>
	<li>Place the rest of the PNS fraction at the bottom of centrifuge tube (11 x 60 mm) and overlay with lipid droplet wash buffer (~3 mL; 4 mL total) (50 mM potassium phosphate buffer pH 7.4, 100 mM potassium chloride, 1 mM EDTA, and 1 mM phenylmethanesulfonyl fluoride).</li>
	<li>Centrifuge for 2 h at 100,000 x g and 4 &#176;C. Harvest the floating lipid droplet fraction using a bent, blunt cannula from the top of the tube (approximately 250 - 500 &#181;L, depending on the amount of lipid droplets). Place the lipid droplet fraction in a centrifuge tube (11 x 60 mm), overlay with lipid droplet wash buffer (~3.5 mL; 4 mL total), and repeat the centrifugation step.</li>
	<li>Harvest the floating lipid droplet fraction using a bent, blunt cannula from the top of the tube and transfer the lipid droplets to a new 1.5 mL microfuge tube. Add 500 &#181;L of lipid droplet wash buffer and spin for 20 min at 21,000 x g and 4 &#176;C. 
	NOTE: Lipid droplets appear as a white band floating on top of the buffer.</li>
	<li>Remove the subjacent wash buffer using a gel loading pipette tip and repeat this washing step three times.</li>
	<li>After the final removal of the washing buffer using a gel loading pipette&#160;tip, mix 5 &#181;L of lipid droplet fractions with 10 &#181;L of NP-40 lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1% NP-40, supplemented with protease inhibitor cocktail). Incubate the sample on ice for 1 h to inactivate the virus if working with infectious material. Determine the protein level with a detergent-compatible protein assay; at least 35 &#181;g of protein is needed for MS-analysis.</li>
	<li>Store the lipid droplet fractions at -20 &#176;C.</li>
	<li>Mix the corresponding volume of the lipid droplet fraction with 4x loading dye. Incubate on ice for 1 h to inactivate the virus if working with infectious material. Boil at 95 &#176;C for 5 min.</li>
	<li>Separate the proteins using SDS-PAGE according to the manufacturers&#39; protocol. Transfer the gel into colloidal Coomassie staining solution. Excise all the protein bands from the gel. After tryptic in-gel digestion24, evaporate the samples and dissolve them in 0.1% formic acid. Analyze by LC-MS/MS. 
	NOTE: Here, LC-MS/MS analyses were performed on a Quadrupole-Time-of-Flight mass spectrometer (Q-TOF) or on a Linear Trap Quadrupole (LTQ) orbitrap mass spectrometer. Both instruments were coupled with an ESI-source to a nano-UPLC system. Data analyses and LC-MS/MS analyses on the Q-TOF and on the orbitrap mass spectrometer were performed as described21,22. Take great care not to contaminate the sample with human keratin. Always use keratin-free pipette tips and tubes. Prepare buffers under the laminar flow hood with keratin-free chemicals. Before usage, clean all surfaces and devices with distilled water and ethanol. Always wear gloves and a lab coat.</li>
</ol>
4. Analysis of Lipid Droplet Purity
<ol>
	<li>Dilute aliquots of lipid droplet fractions 1:2 and input fractions (see step 3.9) 1:10 with NP-40 lysis buffer. Incubate the samples on ice for 1 h to inactivate the virus if working with infectious material. Determine the protein level with a detergent-compatible protein assay.</li>
	<li>Mix equal amounts of protein with 6x sample buffer and incubate the samples on ice for 1 h to inactivate the virus if working with infectious material. Proceed with SDS-PAGE and transfer the proteins onto a nitrocellulose membrane by tank blotting at 80 V for 90 min. 
	NOTE: After blocking the membrane in blocking buffer (5% nonfat dried milk powder in TBS-T (10 mM Tris-HCl pH 7.6, 150 mM NaCl, and 0.05% Tween20)), incubate with antibodies directed against lipid droplet markers (e.g.,&#160;PLIN1, PLIN2, or PLIN3) and markers of other subcellular compartments (e.g.,&#160;CALR, MnSOD, or tubulin), followed by secondary HRP-coupled antibodies. Use chemiluminescence for the detection of proteins.</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a protocol to isolate lipid droplets for quantitative lipid droplet proteome analysis to compare the enrichment and depletion of proteins associated with lipid droplets under diverse culture conditions, such as viral infections. As an alternative method, the proteome analysis can be performed with label-free quantifications based on total peak intensities. This method has no dynamic range limitation and avoids metabolic problems. The advantage of the SILAC approach is that the samples are pooled prior l...

Lipid droplets are highly dynamic cytoplasmic (and nuclear) cell organelles composed of a core of neutral lipids (triglycerides (TG) and cholesterol ester (CE)) enclosed by a monolayer of phospholipids with embedded proteins1. All cell types produce lipid droplets, but they vary in size, lipid composition, and protein decoration. Lipid droplets fulfill diverse functions, including serving as energy and membrane precursor reservoirs or as protein deposits. In addition, through the uptake of lipids, they protect cells from lipotoxicity, release lipids as signaling molecules, and are involved in protein degradation and endoplasmic reticulum (ER) stress responses2. As such, a host of proteins bind to lipid droplets and govern their generation, degradation, trafficking, and interaction with other organelles. Among them are the perilipin family of bona fide lipid droplet binding proteins (PLIN1-5)3.
Lipid droplet biogenesis likely starts at the ER, where ER-resident enzymes catalyze the synthesis of neutral lipids that accumulate within the membrane bilayer, forming a lens of neutral lipids, a process that was recently visualized nicely in yeast4. Membrane bending and elevated phosphatidic acid and diacylglycerol levels are then thought to attract proteins involved in phospholipid biosynthesis, as the simultaneous synthesis of the core neutral lipids and the shielding phospholipids is required for lipid droplet generation5. Enzymes harboring transmembrane domains that reside at the ER catalyze this process. Expansion to large lipid droplets requires the activity of a different class of lipid-synthesizing enzymes that harbor an amphipathic helix and can thus travel from the ER to lipid droplets. The mobilization of lipids from lipid droplets occurs through the local activation of the triglyceride and diacylglycerol lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) or by different autophagic pathways, such as macro- and microlipophagy or chaperone-mediated autophagy6. Lipid droplets interact with other cellular organelles, such as mitochondria (for beta-oxidation and lipid synthesis) and ER (for lipid synthesis and protein trafficking), but also with lysosomes, endosomes, and the vacuoles induced by intracellular bacteria7. Indeed bacteria, viruses, and even parasites target lipid droplets for replication and persistence, among them HCV8.
HCV infection is one of the leading causes of liver-related morbidity and mortality worldwide, accounting for approximately 0.5 million deaths per year9. The true number of HCV infections is unknown, but recent estimates suggest that 130 - 150 million people are chronically infected. No vaccine exists, but the recently approved direct-acting antivirals dramatically increase therapeutic responses compared to the standard interferon-based therapy. However, worldwide, the treatment of patients will likely be restricted due to the extremely high costs of the new therapeutics. About half of all individuals chronically infected with HCV develop fatty liver disease (steatosis), a condition characterized by the excessive accumulation of lipid droplets in hepatocytes. Intriguingly, lipid droplets also emerged as vital cellular organelles for HCV replication, putatively serving as viral assembly sites10,11.
In HCV-infected cells, the viral protein core and NS5A localize to lipid droplets in a process that depends on triglyceride biosynthesis, as inhibitors of diacylglycerol acyltransferase-1 (DGAT1) impair trafficking to lipid droplets and subsequent HCV particle production12,13,14,15. In addition, mutations in the lipid droplet-binding domains of either core or NS5A suppress HCV assembly16,17. Core and NS5A then recruit all other viral proteins, as well as viral RNA replication complexes, to membranes closely associated with lipid droplets16. A concerted action of all viral proteins is required for the successful production of infectious viral progeny10,11. The structural proteins are part of the virions, and the nonstructural proteins promote the protein-protein interactions required for this process. Intriguingly, the bona fide lipid droplet-binding protein PLIN3/TIP47 is required for both HCV RNA replication and the release of virions18,19,20. Despite these recent advances, the mechanistic details, especially of virus-host interactions during the late stages of HCV replication, remain ill-defined, and the precise function of the lipid droplets is unknown.
Here, we describe a method to isolate lipid droplets for the quantitative mass spectrometry of associated proteins. Using this method, we found profound changes in the lipid droplet proteome during HCV infection and identified annexin A3 as a host protein that co-fractionates with lipid droplets and is required for efficient HCV maturation21.

<table><tbody><tr><td>SILAC Protein Quantitation Kit - DMEM</td><td>Thermo Fisher</td><td>89983&amp;nbsp;</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt; L-Arginine-HCl 50&amp;nbsp;mg</td><td>Thermo Fisher</td><td>88210</td><td></td></tr><tr><td>Roti-Load 1</td><td>Roth GmbH</td><td>K929.1</td><td></td></tr><tr><td>Roti-Blue 5x Concentrate</td><td>Roth GmbH</td><td>A152.2&amp;nbsp;</td><td></td></tr><tr><td>10x SDS-Tris-Glycine - Buffer</td><td>Geyer Th. GmbH &amp;amp; Co.KG&amp;nbsp;</td><td>A1415,0250&amp;nbsp;</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Life Technologies GmbH&amp;nbsp;</td><td>350500038&amp;nbsp;</td><td></td></tr><tr><td>Penicillin/Streptomycin Solution for Cell Culture</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>P4333-100ml&amp;nbsp;</td><td></td></tr><tr><td>DPBS 1x Dulbecco&#039;s Phosphate-buffered Saline</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>D8537&amp;nbsp;</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>T3924-100ML&amp;nbsp;</td><td></td></tr><tr><td>Sodium Chloride &lt;em&gt;BioChemica&lt;/em&gt;</td><td>AppliChem GmbH</td><td>A1149,1000</td><td></td></tr><tr><td>Tris Ultrapure&amp;nbsp;&amp;nbsp;</td><td>AppliChem GmbH</td><td>A1086,5000A&amp;nbsp;</td><td></td></tr><tr><td>EDTA &lt;em&gt;BioChemica&lt;/em&gt;</td><td>AppliChem GmbH</td><td>A1103,0250</td><td></td></tr><tr><td>Protease Inhibitor Cocktail 5 mL</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>P8340-5ML&amp;nbsp;</td><td></td></tr><tr><td>D(+)-Sucrose &lt;em&gt;BioChemica&lt;/em&gt;</td><td>AppliChem GmbH</td><td>A3935,1000</td><td></td></tr><tr><td>Hydrochloric acid (HCl) 37% pure Ph. Eur., NF</td><td>AppliChem GmbH</td><td>A0625</td><td></td></tr><tr><td>DC Protein Assay&amp;nbsp;</td><td>Bio-Rad Laboratoris GmbH&amp;nbsp;</td><td>500-0116&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>AppliChem GmbH</td><td>151339</td><td></td></tr><tr><td>SDS Ultrapure</td><td>AppliChem GmbH</td><td>A1112</td><td></td></tr><tr><td>Bromophenol blue</td><td>AppliChem GmbH</td><td>A2331</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol&amp;nbsp;</td><td>AppliChem GmbH</td><td>A4338</td><td></td></tr><tr><td>Blasticidin</td><td>Invivogen</td><td>ant-bl-1&amp;nbsp;</td><td></td></tr><tr><td>Potassium Chloride&amp;nbsp;</td><td>AppliChem GmbH</td><td>A1039</td><td></td></tr><tr><td>Phenylmethanesulfonyl Fluoride&amp;nbsp;</td><td>AppliChem GmbH</td><td>A0999</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>221309</td><td></td></tr><tr><td>Dipotassium Hydrogenphosphate</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>P3786&amp;nbsp;</td><td></td></tr><tr><td>DTT&amp;nbsp;</td><td>AppliChem GmbH</td><td>A2948</td><td></td></tr><tr><td>NP-40</td><td>AppliChem</td><td>A1694</td><td></td></tr><tr><td>TWEEN 20</td><td>AppliChem</td><td>A4974</td><td></td></tr><tr><td>Nonfat dried milk powder</td><td>AppliChem</td><td>A0830</td><td></td></tr><tr><td>Anti-ADFP/ ADRP&amp;nbsp;</td><td>abcam</td><td>ab52355</td><td></td></tr><tr><td>M6PRB1/TIP47 100 &amp;micro;g</td><td>abcam</td><td>ab47639</td><td></td></tr><tr><td>Calreticulin, pAb 200 &amp;micro;g</td><td>Enzo Life Science GmbH&amp;nbsp;</td><td>ADI-SPA-600-F&amp;nbsp;</td><td></td></tr><tr><td>Anti-&amp;beta;-Tubulin&amp;nbsp;</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>T6074 200&amp;micro;l &amp;nbsp;</td><td></td></tr><tr><td>Ethanol absolute (EtOH)</td><td>Geyer Th. GmbH &amp;amp; Co.KG&amp;nbsp;</td><td>A3678,0250 &amp;nbsp;</td><td></td></tr><tr><td>Anti-MnSOD</td><td>Enzo Life Science GmbH&amp;nbsp;</td><td>ADI-SOD-110-F</td><td></td></tr><tr><td>Anti-mouse HRP</td><td>Thermo Fisher Pierce</td><td>32430</td><td></td></tr><tr><td>Anti-rabbit HRP</td><td>Thermo Fisher&amp;nbsp; Pierce</td><td>32460</td><td></td></tr><tr><td>Amersham Hyperfilm ECL</td><td>GE Healthcare</td><td>28906836</td><td></td></tr><tr><td>Lumi-Light Western Blotting Substrate</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>12015196001</td><td></td></tr><tr><td>96-Well Cell Culture Plate</td><td>Greiner Bio-One GmbH&amp;nbsp;</td><td>655 180&amp;nbsp;</td><td></td></tr><tr><td>Terumo Syringe 1 mL</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Filtropur BT 50, 500 mL, 0.45 &amp;micro;m&amp;nbsp;</td><td>SARSTEDT&amp;nbsp;</td><td>83.1823.100&amp;nbsp;</td><td></td></tr><tr><td>Mini-PROTEAN TGX Precast Gels, Any kD resolving gel</td><td>Bio-Rad Laboratoris GmbH&amp;nbsp;</td><td>456-9034&amp;nbsp;</td><td></td></tr><tr><td>6-Well Cell Culture Plate</td><td>Greiner Bio-One GmbH&amp;nbsp;</td><td>657160&amp;nbsp;</td><td></td></tr><tr><td>Dishes Nunclon 150/20&amp;nbsp;</td><td>Fisher Scientific GmbH&amp;nbsp;</td><td>10098720 - 168381&amp;nbsp;</td><td></td></tr><tr><td>Cell Scraper</td><td>neoLab Migge GmbH&amp;nbsp;</td><td>C-8120&amp;nbsp;</td><td></td></tr><tr><td>Tube, 50 mL</td><td>Greiner Bio-One GmbH&amp;nbsp;</td><td>227261&amp;nbsp;</td><td></td></tr><tr><td>SafeSeal Tube RNase-free&amp;nbsp;</td><td>SARSTEDT&amp;nbsp;</td><td>72.706.400&amp;nbsp;</td><td></td></tr><tr><td>Ultra Clear Centrifuge Tubes 11 x 60 mm</td><td>Beckman Coulter GmbH&amp;nbsp;</td><td>344062&amp;nbsp;</td><td></td></tr><tr><td>Suction Needles</td><td>Transcodent</td><td>6482</td><td></td></tr><tr><td>Biosphere Fil. Tip 1000&amp;nbsp;</td><td>SARSTEDT&amp;nbsp;</td><td>70.762.211&amp;nbsp;</td><td></td></tr><tr><td>Biosphere Fil. Tip 200</td><td>SARSTEDT&amp;nbsp;</td><td>70.760.211&amp;nbsp;</td><td></td></tr><tr><td>Biosphere Fil. Tip 10</td><td>SARSTEDT&amp;nbsp;</td><td>70.1130.210&amp;nbsp;</td><td></td></tr><tr><td>Dounce Tissue Grinder</td><td>Fisher Scientific GmbH&amp;nbsp;</td><td>11883722</td><td></td></tr><tr><td>Pestles For Dounce All-Glass Tissue Grinders</td><td>Fisher Scientific GmbH&amp;nbsp;</td><td>10389444</td><td></td></tr><tr><td>Orbitrap Fusion</td><td></td><td></td><td></td></tr><tr><td>Branson Sonifier 450</td><td></td><td></td><td></td></tr><tr><td>Thermomixer comfort, with Thermoblock 1.5 mL</td><td>Eppendorf</td><td>5355&amp;nbsp;000.127</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell, Mini Trans-Blot Module, and PowerPac Basic Power Supply,&amp;nbsp;</td><td>BioRad</td><td>165-8033</td><td></td></tr><tr><td>Mini-PROTEAN 3 Multi-Casting Chamber</td><td>BioRad</td><td>165-4110</td><td></td></tr><tr><td>PowerPac HC Power Supply</td><td>Biorad</td><td>164-5052</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Optima L-90K</td><td>Beckman Coulter GmbH&amp;nbsp;</td><td>365670</td><td></td></tr><tr><td>SW 60 Ti Rotor</td><td>Beckman Coulter GmbH&amp;nbsp;</td><td>335649</td><td></td></tr><tr><td>Infinite M1000 PRO</td><td>Tecan</td><td></td><td></td></tr></tbody></table>

lipid droplet isolation for quantitative mass spectrometry analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are vital to HCV&#160;infection as the putative sites of virion assembly, but the molecular mechanisms of morphogenesis and egress of virions are largely unknown. To identify novel host dependency factors involved in that process, we performed quantitative lipid droplet proteome analysis of HCV-infected cells21 (Figure 1A). We established a protocol for purifying lipid droplets and routinely detected a strong enrichment of the lipid ...

Watch this Scientific Journal Video about Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis at JoVE.com

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

lipid-droplet-isolation-for-quantitative-mass-spectrometry-analysis

Research

JoVE Journal

Biochemistry

10.0K Views.  Leibniz Institute for Experimental Virology. Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Video: Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Chemistry

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Bioengineering

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Biology

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Screening Lipid Structures Using Stable Isotope Labeling

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (&lt;1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Quantification of Complex Lipidomic Samples Using Stable Isotope Labeling

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

Neuroscience

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

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Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit