AM is funded by the Young Group Leader Program (ATIP/Avenir Inserm-CNRS), by la Ligue Nationale contre le Cancer and la Fondation ARC pour la recherche sur le Cancer. We thank Mariette Matondo from the Mass Spectrometry for Biology platform (UTECHS MSBIO, Pasteur Institute, Paris). We thank Lauren Anderson for her reading of the manuscript.

Human blood samples (LRSC) from healthy, de-identified donors were obtained from EFS (French National Blood Service) as part of an authorized protocol (CODECOH DC-2018&#8211;3114). Donors gave signed consent for the use of blood.
1. Media and Buffer Preparation
<ol>
	<li>Prepare the macrophage medium [RPMI glutamax + 10 mM HEPES + 1x non-essential amino acids (NEAA)] and warm it to 37 &#176;C.</li>
	<li>Prepare the macrophage medium + 10% human serum from AB plasma (SAB), filter it (0.22 &#181;m filter), then warm it to 37 &#176;C (referred to as macrophage medium + 10% SAB hereafter).</li>
	<li>Prepare the sorting buffer [1x phosphate buffered saline (PBS) + 0.5% bovine serum albumin (BSA) + 2 mM ethylenediaminetetraacetic acid (EDTA)], filter it (0.22 &#181;m filter), and maintain it at 4 &#176;C.</li>
</ol>
2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Leukoreduction System Chamber (LRSC)
<ol>
	<li>Put 15 mL of density gradient cell separation solution (see Table of Materials) in a 50 mL centrifugation tube so it can warm to room temperature (RT) before receiving the LRSC. 
	NOTE: Density depends on temperature. As this product is stored at 4 &#176;C, this step must be done in advance so it can equilibrate to RT.</li>
	<li>Empty the LRSC into a 50 mL centrifugation tube, add up to 50 mL of 1x PBS, and mix. Very slowly, add 25 mL of the mix prepared during step 2.2 on top of 15 mL of density gradient solution warmed up during step 2.1. 
	NOTE: Be careful not to mix the phases during this step. The blood must be added on the density gradient solution without any disturbance of this phase.</li>
	<li>Centrifuge both centrifugation tubes for 25 min at 700 x g without breaks. 
	NOTE: At the end of the density gradient centrifugation, the layers from bottom to top are: the erythrocytes and granulocytes forming the pellet, density gradient solution phase, layer of PBMCs, and plasma.</li>
	<li>With a pipet, pass through the plasma phase without aspirating it and collect the PBMC layer into a new 50 mL centrifugation tube. Add up to 50 mL of 1x PBS to the PBMCs as a washing step and centrifuge for 10 min at 300 x g.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 40 mL of macrophage medium.</li>
</ol>
3. Magnetic Labeling and Isolation of CD14+ Cells (Monocytes)
<ol>
	<li>Count the PBMCs in a Malassez chamber. Withdraw the amount of PBMCs necessary for conducting the experiment (typically 100 to 300 x 106 cells), place them in a centrifugation tube, and centrifuge for 10 min at 300 x g.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 80 &#181;L of the sorting buffer prepared during step 1.3 per 107 PBMCs. Add 20 &#181;L of CD14 microbeads per 107 PBMCs. Mix well and incubate for 15 min at 4 &#176;C under constant agitation.</li>
	<li>Add 1 mL of sorting buffer per 107 PBMCs as a washing step and centrifuge for 10 min at 300 x g. Aspirate the supernatant and resuspend the pellet in 500 &#181;L of sorting buffer per 108 PBMCs.</li>
	<li>Place a column in the magnetic field of the separator. Prepare the column by rinsing it with 3 mL of sorting buffer.</li>
	<li>Apply the cell suspension onto the column. The column depends on the number of cells to be isolated (here, LS columns for up to 109 PBMCs are used). Collect flow-through containing unlabeled cells. 
	NOTE: Starting at this step, all the tubes (negative and positive selections) are kept for later checking of the different steps by flow cytometry.</li>
	<li>Wash the column with 3 x 3 mL of sorting buffer. Collect unlabeled cells passing through the same tube from step 3.12. Perform washing steps by adding sorting buffer, be careful not to dry your column. Place a collection tube under the column and remove it from the separator.</li>
	<li>Pipette 5 mL of sorting buffer into the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. To increase purity of the CD14+ cells, the eluted fraction is enriched over a second column.</li>
	<li>Repeat steps 3.4 to 3.7 with a new column.</li>
</ol>
4. Plating of Monocytes
<ol>
	<li>Count the monocytes in a Malassez chamber. Check the purity of the CD14+ cells by flow cytometry. Withdraw the amount of monocytes necessary for the experiment and place them in a centrifugation tube.</li>
	<li>Centrifuge for 10 min at 300 x g. Aspirate the supernatant and resuspend the monocyte pellet in macrophage medium. Plate the cells and let them settle for 50 min to 1 h. Aspirate the medium and replace it with macrophage medium + 10% SAB + 25 ng/mL macrophage colony stimulating factor (M-CSF) to induce differentiation.</li>
</ol>
5. Polarization of Macrophages at Day 6
<ol>
	<li>Aspirate the medium. Replace it with macrophage medium + 10% SAB with various stimuli. For example, add 10 ng/mL interferon gamma (INF&#947;) + 1 ng/mL lipopolysaccharide (LPS) to obtain M1 polarization, or 20 ng/mL interleukin 4 (IL4) + 20 ng/mL interleukin 13 (IL13) for M2 polarization. 
	NOTE: The stimulation can be performed between 24 and 48 h before proceeding to other tests.</li>
	<li>Harvest cells using a detaching solution or a cell scraper.</li>
</ol>
6. Cell Culture Under Low Oxygen Conditions
<ol>
	<li>Starting from step 4, maintain the monocytes and then macrophages in an oxygen-controlled environment to perform hypoxic condition analysis. Use a hypoxia working station in order to maintain cells under the desired oxygen partial pressure during the experiment. 
	NOTE: When working under low oxygen pressure, it is important to prepare all media and washing buffers under the station and wait sufficiently to obtain the correct partial pressure in the liquid. For example, 10 mL of PBS in a 60 mm Petri dish requires roughly 2 h to reach 25 mmHg for O2 partial pressure starting from atmospheric pressures (as we have measured it using a fiber-optic oxygen sensor). In many hypoxic stations or incubators, the oxygen pressure is set as a percentage of the atmospheric pressure. If precise measurements are necessary, it is better to use a station authorizing to directly set the oxygen pressure in mmHg.</li>
</ol>
7. Lysis and In-Gel Digestion (Protocol 1)
NOTE: In this and the following sections, two protocols used to obtain peptides and perform LC-MS/MS analysis are described. Protocol 1 describes cell lysis and in-gel fractionation and digestion, and protocol 2 describes in-solution cell lysis followed by in-solution digestion and fractionation using an isoelectric focusing method.
<ol>
	<li>Perform cell lysis in Laemmli buffer [234 mM Tris-HCL (pH 6.8), 7.5% SDS, 37% glycerol, 33.3% (v/v) &#946;-mercaptoethanol, bromophenol blue 0.2% w/v]. Load the protein equivalent of 300,000 cells for each sample on 4-12% bis-Tris acrylamide gels.</li>
	<li>Control the duration of the electrophoretic migration to allow each protein sample to be split into 6 gel bands as exemplified in Figure 3.</li>
	<li>Fix the gel with a fixing solution (30% ethanol + 7.5% acetic acid for 20 min), then add the staining solution (R-250 Coomassie blue for 45 min). Add the destaining solution (30% ethanol + 7.5% acetic until bands appear) before excising the protein bands with a clean scalpel.</li>
	<li>Dice each excised band before introduction in 500 &#181;L tubes. A clean glass surface is warranted to avoid contamination with keratins (5% SDS solution in deionized water can be used to clean surfaces).</li>
	<li>Wash the gel slices 3 times in 200 &#181;L of 25 mM ammonium bicarbonate for 20 min at 37 &#176;C, followed by one wash in 25 mM ammonium bicarbonate and acetonitrile (50% v/v). Dehydrate the gel pieces with 200 &#181;L of 100% acetonitrile for 10 min.</li>
	<li>Incubate each gel piece with 10 mM DTT (dithiothreitol) in 25 mM ammonium bicarbonate for 45 min at 56 &#176;C (200 &#181;L), followed by 55 mM iodoacetamide in 25 mM ammonium bicarbonate (200 &#181;L) for 35 min in the dark at RT.</li>
	<li>To stop alkylation, incubate each gel piece with 200 &#181;L of 10 mM DTT in 25 mM ammonium bicarbonate for 10 min at RT. Wash the gel pieces in 200 &#181;L of 25 mM ammonium bicarbonate, then dehydrate with 200 &#181;L of 100% acetonitrile for 10 min.</li>
	<li>Digest the proteins overnight at 37 &#176;C with Trypsin/Lys-C mix according to the manufacturer&#8217;s instructions.</li>
	<li>Extract the resulting peptides from gel pieces by adding 50 &#956;L of 50% acetonitrile for 15 min, then 50 &#956;L of 5% formic acid for 15 min, and finally, 50 &#956;L of 100% acetonitrile for 15 min. Pool and dry each fraction in low-absorption tubes to limit adsorption of peptides and sample loss. Store the samples at -80 &#176;C until further analysis.</li>
</ol>
8. Protein Extraction and In-Solution Digestion (Protocol 2)
<ol>
	<li>Perform cell lysis (2 x 106 cells) with 150 &#181;L of the following lysis buffer:
	<ol>
		<li>7 M urea, 2 M thiourea, 40 mM Tris, and 4% CHAPS, supplemented with protease inhibitors (complete mini, EDTA-free protease inhibitor cocktail).</li>
	</ol>
	</li>
	<li>Homogenize the solutions for 30 min at RT with a thermoshaker. Centrifuge at 13,800 x g for 20 min RT and keep the supernatant.</li>
	<li>Remove contaminants with a 2D clean-up kit:
	<ol>
		<li>The kit contains precipitant solution, co-precipitant solution, wash buffer, and wash additive.</li>
		<li>Add 300 &#181;L of precipitant solution and mix well. Incubate on ice for 15 min. Add 300 &#181;L of co-precipitant solution. Centrifuge the tubes (at least) at 12,000 x g for 5 min. A small pellet should be visible. Proceed rapidly to the next step to avoid resuspension or dispersion of the pellet. Remove the supernatant without disturbing the pellet.</li>
		<li>Centrifuge the tubes again with the cap-hinge and pellet facing outward to bring any remaining liquid to the bottom of the tube. A brief pulse is sufficient. There should be no visible liquid remaining in the tubes.</li>
		<li>Without disturbing the pellet, add 40 &#181;L of co-precipitant solution. Let the tube sit on ice for 5 min. Centrifuge for 5 min, then remove and discard the wash. Add 25 &#181;L of de-ionized water. Vortex each tube for 5-10 s. The pellet should disperse but not dissolve in the water.</li>
		<li>Add 1 mL of wash buffer (pre-chilled for at least 1 h at -20&#176;C) and 5 &#181;L of wash additive. Vortex until the pellet is fully dispersed. Incubate the tubes at -20&#176;C for at least 30 min. Vortex for 20-30 s every 10 min 
		NOTE: The tubes can be stored at -20&#176;C for up to 1 week with minimal protein degradation or modification.</li>
		<li>Centrifuge the tubes (at least) at 12,000 &#215; g for 5 min. Carefully remove and discard the supernatant. A white pellet should be visible. Allow the pellet to airdry for no more than 5 min (if the pellet is too dry, it will be difficult to resuspend).</li>
	</ol>
	</li>
	<li>Resuspend the protein pellet in 300 &#181;L of 8 M urea and 0.1 M ammonium bicarbonate. Vortex strongly for 1 min. Determine the protein concentration using a colorimetric assay.</li>
</ol>
9. In-Solution Digestion (Protocol 2)
<ol>
	<li>Reduce disulfide bridges by adding 5.1 &#181;L of a 700 mM DTT aqueous solution (final concentration 12.5 mM) to the resuspended proteins from step 8.4 and incubate at 37 &#176;C for 30 min with a thermoshaker. Alkylate cysteine residues by adding 20.3 &#181;L of a 700 mM iodoacetamide aqueous solution (final concentration 40 mM) and incubating at 25&#176;C for 30 min in the dark with a thermoshaker.</li>
	<li>Add 990 &#181;L of 0.1 M ammonium bicarbonate to the sample. Add a corresponding volume of Trypsin/Lys-C mix (enzyme:substrate ratio 1:100 w/w). Incubate at 37&#176;C overnight with a thermoshaker.</li>
</ol>
10. Clean-up Cartridge (Protocol 2)
<ol>
	<li>Wet a cartridge with 1 column-volume (1 mL) of methanol. Clean the cartridge with 1 column-volume (1 mL) of 80% acetonitrile/HPLC-grade water and discard the flow-through. Equilibrate the cartridge with 4 column-volumes (4 mL) of 0.1% formic acid/HPLC-grade water and discard the flow-through.</li>
	<li>Acidify samples with 90 &#181;L of 10% formic acid or water to pH 2-3 (check the pH with a pH indicator). Load the acidified samples and collect the flow-through. Reload the flow-through (containing the not-retained peptides). Wash the cartridge with 6 column-volume (6 mL) of 0.1% formic acid/HPLC-grade water.</li>
	<li>Elute peptides from the cartridge with 1 column-volumes (1 mL) of 0.1% formic acid/50% acetonitrile/HPLC-grade water. Transfer to a 1.5 mL microcentrifuge tube. Concentrate the sample using a vacuum concentrator (150 x g, vacuum at 160 mBar).</li>
</ol>
11. Fractionation by Isoelectric Focusing (Protocol 2)
NOTE: Peptides are separated according to their isoelectric points using an off-gel fractionator on a 13 cm strip covering a pH range from 3 to 10. We used the following protocol provided by the supplier (summarized below):
<ol>
	<li>Prepare the following solutions: solution A (600 &#181;L of glycerol solution, 60 &#181;L of OFFGEL buffer, 4.34 mL of ultrapure water) and solution B (1.776 mL of solution A and 444 &#181;L of ultrapure water).</li>
	<li>Assemble the IPG strips, frames, and electrodes according to the manufacturer&#8217;s instructions.</li>
	<li>Resuspend the sample with 1.8 mL of solution B. Add 40 &#181;L of solution B into each well. Load 150 &#181;L of sample into each well.</li>
	<li>Select the default method for peptides: OG12PE00 (OFFGEL default method for peptides for use with a 3100 OFFGEL Low Res Kit, pH 3-10, 12-well frames. Wait until this method has been completed (~20 h). Collect the fractions in properly labeled tubes.</li>
</ol>
12. Clean-up Harvard Apparatus Column Reverse C18 Post-IEF (Protocol 2)
<ol>
	<li>Progressively add a few &#956;L at a time of 1% TFA in de-ionized water to each fraction to acidify the sample. Check using pH paper that the pH is about 3 or below.</li>
	<li>Prepare the following solutions: solution 1 (5 mL of acetonitrile, 10 &#181;L of formic acid, 4.99 mL of ultrapure water) and solution 2 (0.5 mL of acetonitrile, 10 &#181;L of formic acid, 9.49 mL of ultrapure water).</li>
	<li>Pre-wet the spin column with 150 &#181;L of solution 1. Centrifuge for 90 s at 750 x g and discard the flow-through. Wash the spin column with 150 &#956;L of solution 2. Centrifuge for 90 s at 750 x g and discard the flow-through.</li>
	<li>Pass the fraction through the column. Centrifuge for 90 s at 750 x g and discard the flow-through. Wash with 150 &#956;L of solution 2. Centrifuge for 90 s at 750 x g and discard the flow-through.</li>
	<li>Elute the fraction with 50 &#956;L of solution 1. Centrifuge for 90 s at 750 x g. Repeat these steps once more.</li>
	<li>Dry-fraction using a vacuum concentrator (150 x g, vacuum 160 mBar) and store at -80&#176;C</li>
</ol>
13. Analysis of Proteomic Data and Bioinformatics18
<ol>
	<li>Analyze data obtained by a nano-LC MS/MS mass spectrometer using quantification software such as MaxQuant (version 1.5.2.8) and the Andromeda search engine.</li>
	<li>Set false discovery rate (FDR) to 1% for both proteins and peptides and a minimum length of 7 amino acids. Set enzyme specificity as C-terminal to Arg and Lys. Allow 2 missed cleavages at proline bonds. Select carbamidomethylation of cysteine as a fixed modification and N-terminal protein acetylation and methionine oxidation as variable modifications.</li>
	<li>Further analyze the data with statistical analysis software. Perform a functional enrichment analysis using FunRich software (www.funrich.org/). Perform a gene ontology enrichment analysis using DAVID software (https://david.ncifcrf.gov/).</li>
</ol>

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The authors declare no conflicts of interest.

Because proteomics is a powerful tool to study the expression of different proteins from a whole cell or subcellular compartments, optimization of the cell lysis protocol and digestion of proteins has been addressed by a number of studies. There are three main classes of methods, which include in-gel digestion (digestion of proteins in polyacrylamide gel matrix)17, digestion in solution18 and filter-aided sample preparation19. This last method, at fi...

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis, including removal of apoptotic cells and remodelling of the extracellular matrix1. These cells are characterized by a strong phenotypic plasticity2 that translates into a many possible activation states, which are also called polarizations. The precise molecular description of these various polarizations is a priority in the field of macrophage biology3. It has been proposed to classify these polarizations using the so-called M1/M2 dichotomy, in which M1 represents pro-inflammatory and M2 represents anti-inflammatory macrophages. This model fits well in various pathological situations like acute infections, allergy, and obesity4. However, in chronically inflamed tissues and cancer, it has been demonstrated that this classification is unable to grasp the broad phenotypic repertoire that macrophages present in certain cellular environments5,6,7. The current consensus is that macrophage polarization is better described using a multidimensional model to integrate specific microenvironmental signals8. This conclusion has been confirmed through transcriptomic analysis of human macrophages showing that the M1/M2 model is inefficient in describing the obtained polarizations9.
The study presented aims to provide a protocol to obtain proteomic signatures of various polarizations in human macrophages. We describe how to differentiate human macrophages in environments of various oxygen levels and obtain peptides from the whole macrophage proteome to perform a label-free quantification. This quantification allows the comparison of expression levels of various proteins. As research on stem cells has revealed the importance of oxygen as an environmental key parameter10, we seek to understand how this tissue parameter can influence macrophage polarization in humans. The partial pressure of oxygen has been found to range from 3 to 20% (of total atmospheric pressure) in the human body, where 20% corresponds roughly to what is commonly used in a cell culture incubator (the exact value is around 18.6% while taking the presence of water into account).
Previous work has shown that alveolar differ from interstitial macrophages from functional and morphological point of views11 and that these differences are probably partially due to the different oxygen levels to which they are exposed12. Furthermore, bone marrow-derived macrophages show an increased ability to phagocytize bacteria when exposed to a low oxygen environment12. The opposite effect has been found for THP1-differentiated human macrophages13, but these results support the idea that oxygen is a regulator of macrophage biology and that it is necessary to clarify this role at the molecular level in human macrophages. In a previous study, we have applied a proteomics approach to address these issues. By measuring expression levels for thousands of proteins simultaneously, we highlighted the impact of oxygen on polarization and provided a list of new molecular markers. We were also able to relate these findings to some macrophages functions. Notably, we found that the rate of phagocytosis of apoptotic cells was increased in IL4/IL13-polarized macrophages, which was linked to the upregulation of ALOX15 as revealed by the proteomic analysis14. In the present study, we describe how to perform such an analysis.

<table>
	<tbody>
		<tr>
			<td>Hypoxia Working Station</td>
			<td>Oxford Optronix</td>
			<td>Hypoxylab</td>
			<td></td>
		</tr>
		<tr>
			<td>C6 Flow cytometer</td>
			<td>BD</td>
			<td>Accuri C6</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Agilent Technologies</td>
			<td>5188-6435</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid (FA)</td>
			<td>ARISTAR</td>
			<td>450122M</td>
			<td></td>
		</tr>
		<tr>
			<td>R-250 Coomassie blue</td>
			<td>Biorad</td>
			<td>1,610,436</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide, E.Coli (LPS)</td>
			<td>Calbiochem</td>
			<td>437627</td>
			<td></td>
		</tr>
		<tr>
			<td>2D clean-up kit</td>
			<td>GE Healthcare</td>
			<td>80-6484-51</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, glutamax supplement</td>
			<td>Gibco</td>
			<td>61870044</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (NEAA) Solution 100X</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) 1X</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Harvard Apparatus column Reverse C18 micro spin column</td>
			<td>Harvard Apparatus</td>
			<td>74-4601</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5 M, pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Bis-Tris 4-12%</td>
			<td>Life Technologies SAS</td>
			<td>NP0321 BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14 Microbeads human</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separation column LS</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage colony-stimulating factor (M-CSF)</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-485</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin 4 (IL4)</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin 13 (IL13)</td>
			<td>Miltenyi Biotec</td>
			<td>130-112-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (INF&#947;)</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-482</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14-FITC (clone T&#220;K4)</td>
			<td>Miltenyi Biotec</td>
			<td>130-080-701</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSmix Tube Rotator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid (TFA)</td>
			<td>Pierce</td>
			<td>28904</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C Mix</td>
			<td>PROMEGA</td>
			<td>V5073</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Mini, EDTA-free Protease Inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Density Gradient Solution (Histopaque 1077)</td>
			<td>Sigma Aldrich</td>
			<td>10771-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Sigma Aldrich</td>
			<td>A7089-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum from human male AB plasma (SAB)</td>
			<td>Sigma Aldrich</td>
			<td>H4522-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA) solution 30%</td>
			<td>Sigma Aldrich</td>
			<td>A9576-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisma-base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma Aldrich</td>
			<td>114405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS) 20%</td>
			<td>Sigma Aldrich</td>
			<td>5030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>34888</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma Aldrich</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma Aldrich</td>
			<td>57670</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma Aldrich</td>
			<td>T8656</td>
			<td></td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Sigma Aldrich</td>
			<td>C9426</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL sterile plastic pipette</td>
			<td>VWR</td>
			<td>612-1685</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer C Eppendorf</td>
			<td>VWR</td>
			<td>460-0223</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak tC18 reverse phase cartridges, 100 mg</td>
			<td>Waters</td>
			<td>WAT036820</td>
			<td></td>
		</tr>
	</tbody>
</table>

proteomic analysis of human macrophage polarization under a low oxygen environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

Starting from peripheral blood mononuclear cells (PBMCs) obtained by differential centrifugation, the protocol permits the obtaining of a population of CD14+ monocytes with an assessed purity of more than 98% by flow cytometry (Figure 1). These monocytes are secondarily differentiated toward various polarizations (Figure 2). When a fractionation on gel is chosen, the migration on SDS-page gels is adapted to obtain the ...

Watch this Scientific Journal Video about Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment at JoVE.com

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

proteomic-analysis-human-macrophage-polarization-under-low-oxygen

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

7.9K Views.  Team Mechanobiology, Immunity and Cancer, Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309. We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Video: Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

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 ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

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

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

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

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Summary

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Chapters in this video

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

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

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves