Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

The overall goal of this procedure is to elucidate the protein expression profiles of liquid and solid tumors. This is accomplished by first enriching cancer cells from patient derived blood or tissue samples. In the second step, proteins are isolated from the cancer cells and then mixed with a tumor specific susac quantification standard.

In the final step, the protein mixture is digested. Ultimately, mass spectrometry is used to show the protein expression profiles from liquid and solid tumors. This method can help to answer key questions in the oncology and pathology field, such as how can drug targets and mechanisms of therapy resistance be identified?

Though this method can provide insight into biomarker discovery, it can also be applied to other systems such as the investigation of diverse, solid, and liquid cancers. Demonstrating the procedure will be Dr.Hannibal Berger Institute for Pathology and Dr.Christophe Lanz from my laboratory To prepare liquid tumor cells for fluorescent sorting by flow cytometry, begin by Ali quoting one milliliter of the human mononuclear cell sample suspended in PBS plus 2%FBS for the negative control, and one milliliter for each fluorescent dye used in the final stain. Next, incubate the cells with the appropriate antibodies for isolating leukemic cell population and for the compensation controls on ice.

After 30 minutes, wash the cells twice with PBS plus 2%FCS. Then add a cell viability stain to the appropriate tubes and sort the cells according to the schematic. Collecting the samples in IMDM plus 10%FCS to collect solid tumors by laser capture microdissection.

First, use a microtome to cut five to 10 micrometer thick sections from formin fixed paraffin embedded samples. Next, mount the sections on film covered membrane slides and dry the samples at 37 degrees Celsius after one hour. De parize and rehydrate the sections by successive one minute incubations in xylene absolute ethanol, 70%ethanol and water.

Then stain the sections with hematin for 20 seconds. Rinse them with tap water and collect the cells of interest with the laser capture microdissection system to extract protein from the sorted liquid tumor cells. Spin down the sample three times, washing the cells in 500 microliters of cold PBS during the second two centrifugations.

Next, incubate the cells in 40 microliters of lysis buffer per one times 10 to the six cells. After 15 minutes, centrifuge the lysate and transfer the clear lysate containing supernatant into a new reaction tube to extract protein from the micro dissected solid tumor cells. Incubate the tissue sample with 60 microliters of tissue lysis buffer at 72 degrees Celsius.

After 15 minutes, collect the fluid by a short centrifugation and transfer the supernatant into a new reaction tube. Then sonicate the lysate on ice for three minutes, followed by an incubation with 15 microliters who have 20%sodium ESAL sulfate at 99 degrees Celsius in a heating block with 600 RPM of agitation. After one hour, spin down the lysate and transfer the S natin to a new tube.

Next, determine the protein concentrations of the cleared cellular and super SLAC standard, and then mix equal amounts of the tumor cell pieces with super SLAC standard, followed by lithium. Do desal sulfate buffer at 25%of the sample volume and reducing agent at 10%of the sample volume. Then heat the resulting solution in a heating block for 10 minutes at 72 degrees Celsius.

To measure the protein concentration of the solid tumor lysate on a plate reader mix a standard bovine serum albumin dilution solution series, the lysate and the appropriate super SLAC standard with a commercially available protein assay and a 96 well plate. After one minute of shaking, incubate the sample for the appropriate time period and measure the absorbance as indicated by the manufacturer. Then mix equal amounts of clarified lysate and the super SLAC standard with 200 microliters of urea in a filter unit and centrifuge the samples for 30 minutes.

To analyze the tumor cell lysates by liquid chromatography and mass spectrometric analysis. Begin by resolving the peptides of interest in 30 microliters of loading buffer for five minutes in a sonication bath. Then spin down the protein and transfer the clear supernatant into a mass spectrometric autos sampler vial.

Next, inject five microliters of sample per analysis into the nano lc, ms MS system autos, sampler, and concentrate and desalt the peptides online in a reversed phase C 18 preco mounted and vented column setup. Finally, separate the peptides on a reversed phase C 18 micro column in a self-paced nanos spray column. Using a 90 minute gradient of five to 35%acetyl nitrile versus 0.1%aqueous formic acid at 300 nanoliters per minute, then use a nano spray ion source to transfer ient and analyze the peptides.

Using a top 15 data dependent acquisition method, different types of mass spectrometers can be used for analyzing the peptides. Proteomic profiling of liquid and solid tumors from patients is a promising approach for the discovery of new diagnostic and predictive biomarkers. For example, this first figure demonstrates an exemplary gating strategy for isolating CD one 17 stains, leukemic cells from a liquid tumor sample.

Whereas here, a five to 10 micrometer thick section mounted on film covered membrane slides as just demonstrated. As shown a region of interest was then selected manually and excised by laser capture micro dissection, unsupervised cluster analysis of the protein expression profiles of liquid and solid tumor samples can then be performed by mass spectrometry, allowing the clear separation of the different tumor entities according to their distinct protein expression profiles. These data may then ultimately contribute to the establishment of clinically relevant biomarkers.

Implementation of enrichment strategies for post-translational modifications such as phosphorylation or UIC ventilation can also be performed to answer additional questions like, how can dysregulated kinase patterns be identified After its development? This technique path the way for researchers in the field of pathology and oncology to explore the specific biomarkers and signaling pathways in a variety of cancer entities. After watching this video, you should have a good understanding of how to isolate proteins from cancers as subpopulations for analyzing their protein expression patterns and signaling states by mass spectrometry.