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.

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			<td>SILAC Protein Quantitation Kit - DMEM</td>
			<td>Thermo Fisher</td>
			<td>89983&#160;</td>
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			<td>13C6 L-Arginine-HCl 50 mg</td>
			<td>Thermo Fisher</td>
			<td>88210&#160;</td>
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			<td>Roth GmbH</td>
			<td>K929.1</td>
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			<td>Roth GmbH</td>
			<td>A152.2&#160;</td>
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			<td>Geyer Th. GmbH &#38; Co.KG&#160;</td>
			<td>A1415,0250&#160;</td>
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			<td>GlutaMAX (100x)</td>
			<td>Life Technologies GmbH&#160;</td>
			<td>350500038&#160;</td>
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			<td>Penicillin/Streptomycin Solution for Cell Culture</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>P4333-100ml&#160;</td>
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			<td>PBS 1x Dulbeccos Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>D8537&#160;</td>
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			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
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			<td>AppliChem GmbH</td>
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			<td>AppliChem GmbH</td>
			<td>A1086,5000A&#160;</td>
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			<td>EDTA BioChemica</td>
			<td>AppliChem GmbH</td>
			<td>A1103,0250</td>
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			<td>Protease Inhibitor Cocktail 5ml</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>P8340-5ML&#160;</td>
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		<tr>
			<td>D(+)-Sucrose BioChemica</td>
			<td>AppliChem GmbH</td>
			<td>A3935,1000</td>
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			<td>Hydrochloric acid 37% pure Ph. Eur., NF</td>
			<td>AppliChem GmbH</td>
			<td>A0625</td>
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		<tr>
			<td>DC Protein Assay&#160;</td>
			<td>Bio-Rad Laboratoris GmbH&#160;</td>
			<td>500-0116&#160;</td>
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			<td>Glycerol</td>
			<td>AppliChem GmbH</td>
			<td>151339</td>
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			<td>SDS Ultrapure</td>
			<td>AppliChem GmbH</td>
			<td>A1112</td>
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		<tr>
			<td>Bromophenol blue</td>
			<td>AppliChem GmbH</td>
			<td>A2331</td>
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		<tr>
			<td>&#946;-Mercaptoethanol&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A4338</td>
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		<tr>
			<td>Blasticidin</td>
			<td>Invivogen</td>
			<td>ant-bl-1&#160;</td>
		</tr>
		<tr>
			<td>Potassium Chloride&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A1039</td>
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		<tr>
			<td>Phenylmethanesulfonyl Fluoride&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A0999</td>
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		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>221309</td>
		</tr>
		<tr>
			<td>Dipotassium Hydrogenphosphate</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>P3786&#160;</td>
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		<tr>
			<td>DTT&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A2948</td>
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			<td>NP-40</td>
			<td>AppliChem</td>
			<td>A1694</td>
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			<td>TWEEN 20</td>
			<td>AppliChem</td>
			<td>A4974</td>
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			<td>Nonfat dried milk powder</td>
			<td>AppliChem</td>
			<td>A0830</td>
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		<tr>
			<td>Anti-ADFP/ ADRP&#160;</td>
			<td>abcam</td>
			<td>ab52355</td>
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		<tr>
			<td>M6PRB1/TIP47 100&#181;g&#160;</td>
			<td>abcam</td>
			<td>ab47639</td>
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			<td>Calreticulin, pAb 200 &#181;g</td>
			<td>Enzo Life Science GmbH&#160;</td>
			<td>ADI-SPA-600-F&#160;</td>
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		<tr>
			<td>Anti-&#223;-Tubulin&#160;</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>T6074 200&#181;l &#160;</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Geyer Th. GmbH &#38; Co.KG&#160;</td>
			<td>A3678,0250 &#160;</td>
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		<tr>
			<td>Anti-MnSOD</td>
			<td>Enzo Life Science GmbH&#160;</td>
			<td>ADI-SOD-110-F</td>
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		<tr>
			<td>Anti-mouse HRP</td>
			<td>Thermo Fisher Pierce</td>
			<td>32430</td>
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			<td>Anti-rabbit HRP</td>
			<td>Thermo Fisher&#160; Pierce</td>
			<td>32460</td>
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		<tr>
			<td>Amersham Hyperfilm ECL</td>
			<td>GE Healthcare</td>
			<td>28906836</td>
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		<tr>
			<td>Lumi-Light Western Blotting Substrate</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>12015196001</td>
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		<tr>
			<td>96 Well Cell Culture Plate</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>655 180&#160;</td>
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			<td>Terumo Syringe 1 ml</td>
			<td>Terumo</td>
			<td>SS-01T</td>
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			<td>Filtropur BT 50, 500 ml, 0.45 &#181;m&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>83.1823.100&#160;</td>
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		<tr>
			<td>Mini-PROTEAN TGX Precast Gels, Any kD resolving gel</td>
			<td>Bio-Rad Laboratoris GmbH&#160;</td>
			<td>456-9034&#160;</td>
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		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>657160&#160;</td>
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			<td>Dishes Nunclon 150/20&#160;</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>10098720 - 168381&#160;</td>
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		<tr>
			<td>Cell Scraper</td>
			<td>neoLab Migge GmbH&#160;</td>
			<td>C-8120&#160;</td>
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			<td>Tube, 50 ml</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>227261&#160;</td>
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			<td>SafeSeal Tube RNase-free&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>72.706.400&#160;</td>
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			<td>Ultra Clear Centrifuge Tubes 11 x 60 mm</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>344062&#160;</td>
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		<tr>
			<td>Suction Needles</td>
			<td>Transcodent</td>
			<td>6482</td>
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		<tr>
			<td>Biosphere Fil. Tip 1000&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>70.762.211&#160;</td>
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		<tr>
			<td>Biosphere Fil. Tip 200</td>
			<td>SARSTEDT&#160;</td>
			<td>70.760.211&#160;</td>
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			<td>Biosphere Fil. Tip 10</td>
			<td>SARSTEDT&#160;</td>
			<td>70.1130.210&#160;</td>
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			<td>Dounce Tissue Grinder</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>11883722</td>
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			<td>Pestles For Dounce All-Glass Tissue Grinders</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>10389444</td>
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		<tr>
			<td>Orbitrap Fusion</td>
			<td></td>
			<td></td>
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			<td>Branson Sonifier 450</td>
			<td></td>
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			<td>Thermomixer comfort, with Thermoblock 1.5 ml</td>
			<td>Eppendorf</td>
			<td>5355&#160;000.127</td>
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		<tr>
			<td>Mini-PROTEAN Tetra Cell, Mini Trans-Blot Module, and PowerPac Basic Power Supply,&#160;</td>
			<td>BioRad</td>
			<td>165-8033</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN 3 Multi-Casting Chamber</td>
			<td>BioRad</td>
			<td>165-4110</td>
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		<tr>
			<td>PowerPac HC Power Supply</td>
			<td>Biorad</td>
			<td>164-5052</td>
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		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424R</td>
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		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424</td>
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		<tr>
			<td>Optima L-90K</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>365670</td>
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		<tr>
			<td>SW 60 Ti Rotor</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>335649</td>
		</tr>
		<tr>
			<td>Infinite M1000 PRO</td>
			<td>Tecan</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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

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

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

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

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

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

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

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

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

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

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

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

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

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

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

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

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

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

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

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

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

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

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

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

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

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

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

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

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