superparamagnetic antibody-based mitochondrial purification

Quantificação aprimorada de proteínas usando isolamento mitocondrial em duas etapas

Mitochondria are complex and dynamic organelles able to sense and adapt to the metabolic needs of the cell. Central to the complexity of cellular metabolism, mitochondria act as metabolic hubs where carbohydrate, protein, lipid, nucleic acid, and co-factor metabolism reactions converge1. They also serve as signaling organelles for pathways of the innate immune response and in response to changes in ions and reactive oxygen species2,3. To date, around 1,100 proteins have been mapped to mitochondria4,5,6, yet we can assume that many more remain to be discovered, especially those expressed only in certain cell types or transiently under specific environmental conditions. Developing new approaches for quantifying changes in mitochondrial composition in metabolic states of interest will increase our knowledge of these organelles and highlight novel therapeutic avenues for the disorders characterized by mitochondrial dysfunction7.
Currently, different mitochondria isolation protocols are available, with differing yields and levels of purity8. Centrifugation-based approaches are the most popular, due to their simplicity and low cost. Although suitable for most applications, differential centrifugation has the disadvantage of obtaining lower mitochondrial purity and requiring large amounts of starting material when more complex density gradient-based applications are used. In recent years, new methods for mitochondria isolation have emerged, such as tag-based immune capture (&#34;MITO-IP&#34;)9 and fluorescence-activated organelle sorting10. Although both procedures can generate samples with high purity, the former requires genetic engineering to tag mitochondria for affinity purification, making the protocols incompatible with primary material from unmodified organisms or human donors. Meanwhile, the latter is dependent on access to flow cytometry and sorting instruments. Combining different isolation methods offers the promise of generating more robust protocols and increased purity.
Here, we present a new protocol for mitochondria isolation based on the combination of two existing methods: (1) differential centrifugation to isolate a crude mitochondrial fraction, and (2) tag-free immune capture of mitochondria with superparamagnetic beads covalently bound to antibodies against translocase of outer mitochondrial membrane 22 (Tomm22)11, a ubiquitous mitochondrial outer-membrane protein (Figure 1). The procedure we describe is compatible with quantitative protein mass spectrometry, and because it is tag-free and does not require genetic manipulation, it can be applied to a wide range of research models, from cell lines to body fluids to whole animal tissues. Furthermore, the use of two steps in the protocol enables the use of subtractive proteomics6,12 for the discovery of novel mitochondrial proteins and the study of their expression.

Autor em destaque: Isolamento sem marcação em duas etapas de mitocôndrias para melhor descoberta e quantificação de proteínas  

Isolamento sem etiquetas em duas etapas de mitocôndrias para melhor descoberta e quantificação de proteínas

Our lab studies changes in mitochondria and energy metabolism and how these impact both healthy and pathological processes like immune activation, cancer, or metabolic disorders. Our aim is to help to develop new treatments to a wide variety of disorders by providing a better understanding of mitochondrial function and energy metabolism. Tools such as mass spectrometry based proteomics, metabolomics, and CRISPR-Cas9 genetic screens are becoming more popular for identifying molecular mechanisms in both the mitochondrial function and energy metabolism in specific cell types and environments.In contrast to other methods, our protocol employs two purification steps, differential centrifugation, and immune capture for mitochondrial isolation. This offers several advantages, like improved removal of contaminants, and it is also powerful tool for identifying new mitochondrial proteins. Our protocol provides insight into the composition, and therefore, mitochondrial function in different cell types and environments.This can help us better understand normal processes like immune activation, but also pathological processes like cancer. We will focus on mitochondrial macrophages. We will test how the composition of mitochondria changes during immune response to better understand their function.Our findings could lead to a development of drugs with, for example, anti-inflammatory effects.

Watch this Scientific Journal Video about Superparamagnetic Antibody-Based Mitochondrial Purification at JoVE.com

Superparamagnetic Antibody-Based Mitochondrial Purification  

Purificação mitocondrial baseada em anticorpos superparamagnéticos

Comece transferindo uma fração bruta de mililitro de mitocôndrias RAW 264,7 para um tubo cônico de 15 mililitros. Adicionar 7 mililitros de tampão mitocondrial salgado suplementado com 150 milimolares de cloreto de sódio. À suspensão, adicione 50 microlitros de contas TOM22 e, em seguida, incube o tubo em uma roda giratória em baixa velocidade.Coloque uma coluna no ímã e equilibre a coluna com oito mililitros de tampão mitocondrial salgado. Descarte o fluxo. Após a incubação da amostra, transfira-a para a coluna e descarte o fluxo.Em seguida, lave a coluna três vezes com oito mililitros de tampão mitocondrial salgado. Retire a coluna do ímã e coloque-a em um tubo cônico de 15 mililitros. Para eluir as mitocôndrias, adicione 1,5 mililitros de tampão mitocondrial salgado e aplique imediatamente um êmbolo para eluir a fração purificada no tubo.Transfira a fração eluída para um novo tubo de 1,5 mililitro e realize a centrifugação a 21.000G por 10 minutos a 4 graus Celsius. Descarte o sobrenadante formado e armazene a pastilha mitocondrial marrom pura a 20 graus Celsius para estudos futuros. A análise imunoblot dos extratos mitocondriais purificados mostrou enriquecimento da citrato sintase mitocondrial e depleção de proteínas de outras organelas celulares.Estudos de integridade mitocondrial por microscopia eletrônica revelaram mitocôndrias com forma oval clássica e cristas intactas circundadas por esferas revestidas com anticorpos elétron-densos. Além disso, a análise do proteoma mostrou enriquecimento de proteínas mitocondriais e duas populações distintas correspondentes a proteínas mitocondriais e não mitocondriais.

superparamagnetic-antibody-based-mitochondrial-purification

Research

JoVE Journal

Biology

Superparamagnetic Antibody-Based Mitochondrial Purification

377 visualizações.  University of Lausanne. Comece transferindo uma fração bruta de mililitro de mitocôndrias RAW 264,7 para um tubo cônico de 15 mililitros. Adicionar 7 mililitros de tampão mitocondrial salgado suplementado com 150 milimolares de cloreto de sódio. À suspensão, adicione 50 microlitros de contas TOM22 e, em seguida, incube o tubo em uma roda giratória em baixa velocidade.Coloque uma coluna no ímã e equilibre a coluna com oito mililitros de tampão mitocondrial salgado. Descarte o fluxo. Após a incuba?...

Vídeo: Superparamagnetic Antibody-Based Mitochondrial Purification  

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial ...

Biologia

Crude Mitochondrial Fraction Preparation

Begin by aspirating the media from the plates containing adherent raw 264.7. Add 10 milliliters of PBS per 15 centimeter plate. Use a cell scraper to detach the cells and pull them into a single 50 milliliter tube.Pipette the cell suspension up and down to homogenize. Next, transfer 5%of the cell suspension volume into a new 1.5 milliliter tube. Centrifuge at 300G for five minutes at room temperature.Discard the supernatant and place the total cell fraction pellet on ice. After centrifuging the remaining suspension as described previously, aspirate the supernatant and re-suspend the cell pellet in five milliliters of ice cold mitochondria buffer. Centrifuge the suspension at 300G for five minutes at four degrees Celsius.Re-suspend the pellet in 0.5 milliliters of cold mitochondrial buffer. And transfer the suspension to a 1.5 milliliter tube. Next, homogenize the suspension, bypassing it 30 times through a 25 gauge needle fitted to a one milliliter syringe.Then add one milliliter of cold mitochondria buffer to the tube and mix by inversion. After centrifuging the cells, transfer one milliliter of the supernatant to a fresh tube on ice. Re-suspend the pellet in the buffer and homogenize the pellet by passing through a 25 gauge needle fitted to a one milliliter syringe.Pull the homogenized cell pellets and the supernatant from the previous steps into a single 1.5 milliliter tube. Centrifuge it at 2, 000G for five minutes at four degrees Celsius. Collect the the supernatant and divide it into four 1.5 milliliter tubes.Make up the volume in each tube to 1.5 milliliters using mitochondrial buffer. Invert the tubes to mix their contents and then centrifuge the tubes. Carefully remove the supernatant and the upper white cellular pellet.Keep one of the four tubes containing the brown mitochondrial pellet on ice as a crude mitochondrial fraction. Pull the remaining pellets into a new tube and make the final volume one milliliter with mitochondria buffer. Using this protocol, total cells and crude mitochondrial fractions were obtained from the raw 264.7 macrophage cell line and BMDM.Immuno blood analysis showed that the mitochondrial citrate synthase was enriched in the crude fraction. Proteins from cytosol, plasma membrane, nucleus, lysosome, and endoplasmic reticulum disappeared. Proteome analysis showed an enrichment of more than tenfold in the crude fraction.The other six cellular compartments were depleted during purification.

Preparação da Fração Mitocondrial Bruta

Begin by transferring one milliliter crude fraction of RAW 264.7 mitochondria into a 15-milliliter conical tube. Add 7 milliliters of salted mitochondrial buffer supplemented with 150 millimolar sodium chloride. To the suspension, add 50 microliters of TOM22 beads and then incubate the tube on a rotating wheel at low speed.Place a column on the magnet and equilibrate the column with eight milliliters of salted mitochondrial buffer. Discard the flow-through. After sample incubation, transfer it to the column and discard the flow-through.Next, wash the column three times with eight milliliters of salted mitochondrial buffer. Remove the column from the magnet and place it in a 15-milliliter conical tube. To elute the mitochondria, add 1.5 milliliters of salted mitochondrial buffer and immediately apply a plunger to elute the purified fraction into the tube.Transfer the eluted fraction to a new 1.5-milliliter tube and perform centrifugation at 21, 000G for 10 minutes at 4 degrees Celsius. Discard the supernatant formed and store the brown pure mitochondrial pellet at 20 degrees Celsius for future studies. Immunoblot analysis of the purified mitochondrial extracts showed enrichment of mitochondrial citrate synthase and depletion of proteins from other cell organelles.Mitochondrial integrity studies using electron microscopy revealed mitochondria with a classic oval shape and intact cristae surrounded by electron-dense antibody-coated beads. Further, proteome analysis showed enrichment of mitochondrial proteins and two distinct populations corresponding to mitochondrial and non-mitochondrial proteins.