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 er en mye brukt massespektrometri teknikk i kjemi og biokjemi. Det har også blitt anvendt i medisinen for å identifisere molekyler og biomarkører. Nylig har det vært brukt i mikrobiologi for rutinen identifisering av bakterier vokst fra kliniske prøver, uten forberedelse eller fraksjoneringstrinn. Vi og andre har brukt denne hel-celle MALDI-TOF massespektrometri teknikken med hell til eukaryote celler. Aktuelle søknader spenner fra celletype identifikasjon til kvalitetskontroll vurdering av cellekultur og diagnostiske applikasjoner. Her beskriver vi bruken for å utforske de forskjellige polarisasjons-fenotypene av makrofager som respons på cytokiner eller varmedrepte bakterier. Det tillates identifisering av makrofager spesifikke fingeravtrykk som er representative for mangfoldet av proteomikk svar av makrofager. Denne søknaden illustrerer nøyaktighet og enkelhet ved fremgangsmåten. Protokollen vi beskrevet her kan være nyttig for studere immunrespons i verten patologiske tilstander eller kan bli utvidet til bredere diagnostiske anvendelser.
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 or to monitor post-translational modifications on 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)-γ 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-β1 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.
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</…
The authors have nothing to disclose.
Richard Ouedraogo is supported by a grant from the Ministère de la Santé (PHRC 2010). We thank Laurent Gorvel, Christophe Flaudrops and Nicolas Amstrong for technical assistance.