Richard Ouedraogo is supported by a grant from the Minist&#232;re de la Sant&#233; (PHRC 2010). We thank Laurent Gorvel, Christophe Flaudrops and Nicolas Amstrong for technical assistance.

In the present protocol, the whole-cell MALDI-TOF technique is used to obtain a mass spectrum considered as a fingerprint of the sample. A bioinformatic analysis allowed the comparison and the classification of these fingerprints. There were three main parts in this protocol:
<ol>
	<li>The preparation of the biological samples: control macrophages and macrophages stimulated with different agonists.</li>
	<li>The analysis of each type of sample with technical replicates by whole-cell MALDI-TOF MS.</li>
	<li>The bioinf...

<ol>
	<li>Mass Gross, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spectrometry: A Textbook. , (2011).</li><li>Villanueva, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+Peptide+Profiling+by+Magnetic+Particle-Assisted,+Automated+Sample+Processing+and+MALDI-TOF+Mass+Spectrometry..">Serum Peptide Profiling by Magnetic Particle-Assisted, Automated Sample Processing and MALDI-TOF Mass Spectrometry..</a> Anal. Chem. 76 (6), 1560-1570 (2004).</li><li>Diamandis, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry+as+a+Diagnostic+and+a+Cancer+Biomarker+Discovery+Tool+Opportunities+and+Potential+Limitations.">Mass Spectrometry as a Diagnostic and a Cancer Biomarker Discovery Tool Opportunities and Potential Limitations.</a> Molec. Cell. Proteomics. 3 (4), 367-378 (2004).</li><li>Pusch, W., Kostrzewa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+MALDI-TOF+Mass+Spectrometry+in+Screening+and+Diagnostic.">Application of MALDI-TOF Mass Spectrometry in Screening and Diagnostic.</a> 11 (20), 2577-2591 (2005).</li><li>Aebersold, R., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+proteomics.">Mass spectrometry-based proteomics.</a> Nature. 422 (6928), 198-207 (2003).</li><li>Domon, B., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry+and+Protein+Analysis.">Mass Spectrometry and Protein Analysis.</a> Science. 312 (5771), 212-217 (2006).</li><li>Carbonnelle, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robustness+of+two+MALDI-TOF+mass+spectrometry+systems+for+bacterial+identification.">Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification.</a> J. Microbiol. Methods. 89 (2), 133-136 (2012).</li><li>Bizzini, A., Jaton, K., Romo, D., Bille, J., Prod&#8217;hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-Assisted+Laser+Desorption+Ionization&#8211;Time+of+Flight+Mass+Spectrometry+as+an+Alternative+to+16S+rRNA+Gene+Sequencing+for+Identification+of+Difficult-To-Identify+Bacterial+Strains.">Matrix-Assisted Laser Desorption Ionization&#8211;Time of Flight Mass Spectrometry as an Alternative to 16S rRNA Gene Sequencing for Identification of Difficult-To-Identify Bacterial Strains.</a> J. Clin. Microbiol. 49 (2), 693-696 (2011).</li><li>Buchanan, C. M., Malik, A. S., Cooper, G. J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualisation+of+peptide+hormones+in+cultured+pancreatic+islet+alpha-+and+beta-cells+by+intact-cell+mass+spectrometry.">Direct visualisation of peptide hormones in cultured pancreatic islet alpha- and beta-cells by intact-cell mass spectrometry.</a> Rapid Commun. Mass Spectrom. 21 (21), 3452-3458 (2007).</li><li>Ouedraogo, R., Flaudrops, C., Ben Amara, A., Capo, C., Raoult, D., Mege, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+circulating+immune+cells+by+matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry.">Global analysis of circulating immune cells by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.</a> PloS One. 5 (10), 13691 (2010).</li><li>Munteanu, B., von Reitzenstein, C., H&#228;nsch, G. M., Meyer, B., Hopf, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+robust+and+automated+protein+analysis+of+cell+differentiation+and+of+primary+human+blood+cells+by+intact+cell+MALDI+mass+spectrometry+biotyping.">Sensitive robust and automated protein analysis of cell differentiation and of primary human blood cells by intact cell MALDI mass spectrometry biotyping.</a> Anal. Bioanal. Chem. 404 (8), 2277-2286 (2012).</li><li>Feng, H., Sim, L. C., Wan, C., Wong, N. S. C., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+characterization+of+protein+productivity+and+production+stability+of+CHO+cells+by+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry.">Rapid characterization of protein productivity and production stability of CHO cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.</a> Rapid Commun. Mass Spectrom. 25 (10), 1407-1412 (2011).</li><li>Ouedraogo, R., Daumas, A., Ghigo, E., Capo, C., Mege, J. -. L., Textoris, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+MALDI-TOF+MS:+A+new+tool+to+assess+the+multifaceted+activation+of+macrophages.">Whole-cell MALDI-TOF MS: A new tool to assess the multifaceted activation of macrophages.</a> J. Proteomics. 75 (18), 5523-5532 (2012).</li><li>Amann, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+profiling+of+proteins+in+lung+cancer+cells+from+fine-needle+aspirates+by+matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry.">Selective profiling of proteins in lung cancer cells from fine-needle aspirates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.</a> Clin. Cancer Res. 12 (17), 5142-5150 (2006).</li><li>Maurer, K., Eschrich, K., Schellenberger, W., Bertolini, J., Rupf, S., Remmerbach, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+brush+biopsy+analysis+by+MALDI-ToF+Mass+Spectrometry+for+early+cancer+diagnosis.">Oral brush biopsy analysis by MALDI-ToF Mass Spectrometry for early cancer diagnosis.</a> Oral Oncol. 49 (2), 152-156 (2013).</li><li>Ishii, K. J., Koyama, S., Nakagawa, A., Coban, C., Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+innate+immune+receptors+and+beyond:+making+sense+of+microbial+infections.">Host innate immune receptors and beyond: making sense of microbial infections.</a> Cell Host Microbe. 3 (6), 352-363 (2008).</li><li>Mantovani, A., Sica, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+comes+of+age.">Macrophage polarization comes of age.</a> Immunity. 23 (4), 344-346 (2005).</li><li>Martinez, F. O., Sica, A., Mantovani, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+polarization.">Macrophage activation and polarization.</a> Front. Biosci. 13, 453-461 (2008).</li><li>Mosser, D. M., Edwards, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nat. Rev. Immunol. 8 (12), 958-969 (2008).</li><li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nat. Rev. Immunol. 11 (11), 723-737 (2011).</li><li>Benoit, M., Desnues, B., Mege, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+bacterial+infections.">Macrophage polarization in bacterial infections.</a> J. Immunol. 181 (6), 3733-3739 (2008).</li><li>Biswas, S. K., Sica, A., Lewis, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+macrophage+function+during+tumor+progression:+regulation+by+distinct+molecular+mechanisms.">Plasticity of macrophage function during tumor progression: regulation by distinct molecular mechanisms.</a> J. Immunol. 180 (4), 2011-2017 (2008).</li><li>Cassetta, L., Cassol, E., Poli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+health+and+disease.">Macrophage polarization in health and disease.</a> Sci. World J. 11, 2391-2402 (2011).</li><li>Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages.">Alternative activation of macrophages.</a> Nat. Rev. Immunol. 3 (1), 23-35 (2003).</li><li>Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+system+in+diverse+forms+of+macrophage+activation+and+polarization.">The chemokine system in diverse forms of macrophage activation and polarization.</a> Trends Immunol. 25 (12), 677-686 (2004).</li><li>Delaby, A., Gorvel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+monocyte+dynamics+in+Q+fever+granuloma+deficiency.">Defective monocyte dynamics in Q fever granuloma deficiency.</a> J. Infect. Dis. 205 (7), 1086-1094 (2012).</li><li>Gibb, S., Strimmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDIquant:+a+versatile+R+package+for+the+analysis+of+mass+spectrometry+data.">MALDIquant: a versatile R package for the analysis of mass spectrometry data.</a> Bioinformatics. 28 (17), 2270-2271 (2012).</li></ol>

The authors have no conflict of interest to declare. RO, JLM and CC are inventors of an international patent WO 2011/154650, named "Proc&#233;d&#233; d&#39;identification de cellules de mammif&#232;res par spectrom&#233;trie de masse MALDI-TOF".

This protocol describes the use of MALDI-TOF-MS on eukaryotic whole cells. Here, we illustrate the accuracy of the method by analyzing the multiple activation states of macrophages in response to their microenvironment.
The success of the protocol relies on few critical steps. First, any solution contaminant may alter the spectra. For example, it is important to wash cells in PBS to remove culture medium and serum proteins before deposition on the target. A cell concentration of 1 x 105</...

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) is a popular mass spectrometry technique to study biological samples. Using a laser beam and an energy-absorbing matrix allows a soft ionization process: the evaporation and genesis of large mostly single-charged biomolecules. This process is called desorption/ionization, justifying the acronym MALDI. These ions are then accelerated by application of voltage and enter a TOF analyzer that allows the separation of these ions and the quantification of their respective masses1.
MALDI-TOF MS has been extensively used in biology, chemistry, and medicine to identify molecules and biomarkers2-4&#160;or to monitor post-translational modifications on&#160;proteins5,6. Recently, several groups applied MALDI-TOF MS to the identification of microorganisms from clinical samples7,8. This microbiological application is now used routinely in the clinical settings. Whole cell MALDI-TOF has many advantages compared to classical applications of MALDI-TOF MS. Samples containing whole cells are directly processed, avoiding time consuming steps to fractionate or separate large amounts of material. Moreover, no characterization of the various peaks is needed: the whole spectrum is considered as a fingerprint of the sample, and matching algorithms compare the tested spectrum with a database of reference spectra.
We and others have applied this whole-cell analysis technique to eukaryotic cells. Many applications may be derived from this technique: (1) identify the main cell types from a mixed sample9-11; (2) assess the viability of cell cultures over time (including quality control industrial applications)12; (3) monitor activation states of a single cell type13; (4) assess the malignant transformation of a clinical sample14,15.
Here, we describe the use of whole-cell MALDI-TOF MS to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. Macrophages play a pivotal role in the immune response to microbial pathogens. They detect infectious agents in the tissues through pattern recognition receptors able to detect conserved microbial patterns, such as lipopolysaccharide (LPS)16. Macrophages are professional antigen-presenting cells that interact with T cells to mount the adaptive immune response. T cells influence macrophages by releasing cytokines that either reinforce or regulate the microbicidal activity of macrophages. By analogy to the Th1/Th2 lymphocyte polarization, inflammatory, microbicidal, and tumoricidal macrophages have been classified into M1 macrophages and immunoregulator macrophages as M2 macrophages17-19. The term M1 refers to the classical activation of macrophages by type I cytokines, such as interferon (IFN)-&#947; and tumor necrosis factor (TNF), or bacterial products, such as LPS18,20-23, whereas macrophages activated by alternative pathways (interleukin (IL)-4, IL-10, Transforming Growth Factor-&#946;1 are considered M2 macrophages19,24,25. The high phenotypic and functional plasticity of macrophages in response to their microenvironment renders these macrophages useful to analyze subtle changes by a MALDI-TOF MS approach.

whole-cell maldi-tof mass spectrometry is an accurate and rapid method to analyze different modes of macrophage activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

The aim of the present protocol is to demonstrate the accuracy of whole-cell MALDI-TOF MS to assess the responsiveness of macrophages to their microenvironment.
Figure 1 describe preparation of stimulated macrophages from blood samples. Figure 2 represents the analysis of monocytes and MDMs by flow cytometry. Note that monocytes expressed CD14 but not CD68 (Figure 1A). Conversely, MDMs expressed CD68 but not CD14 (Figure 1B).<...

Watch this Scientific Journal Video about Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation at JoVE.com

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

whole-cell-maldi-tof-mass-spectrometry-is-an-accurate-rapid-method-to

Research

JoVE Journal

Immunology and Infection

8.7K visualizações.  Aix Marseille Université. This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.