This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.
So, this is unique ambient mass spectrometry, particularly suitable for analyzing biological samples, both in vitro and in vivo. As this does not require regularly a special pretreatment of samples, such as cell sorting, fractionation, and concentration, it can analyze a sample with minimum loss of biological components, especially those that exist as very small amount. This technique allows you to analyze the raw tissue materials directly in a minute by mass spectrometry.
Rapidity is unhindering in robustness of system, chief advantages inherent to this technique. All you have to do is to just to prepare silica, 10 milligrams of tissues specimen, and 10 microliters of proliferate. And put them into disposable cartridge.
It is set to the PESI-MS machine, and you'll get the result in a few minutes. By choosing the appropriate mass spectrometer, you can annotate the molecular identity. To begin, retrieve the liver or kidney tissue from the freezer.
On a cork plate, cut the tissue specimen to a approximately two by two by two millimeters using a scalpel or knife. Alternatively, punch out the sample with the Trepan and trim the length of the column to two millimeters. If the tissue is contaminated with blood, wash briefly with ice-cold phosphate-buffered saline.
Place approximately 10 milligrams of the cut or punched out sample into a plastic microtube and add 100 microliters of 50%ethanol. Use a micropestle to homogenize the sample. Then with the pipetman, place 10 microliters of the homogenate into the well of the cartridge of the mass spectrometer.
Place the cartridge in the ionization chamber of the Direct Probe Ionization Mass Spectrometer and install the stainless steal needle probe that will be used for the sample ionization. Close the chamber lid firmly to automatically activate the safety device. Start the on-board computer by clicking the start icon to analyze, using the on-screen panels, apply a voltage of 2.3 kilovolts to the needle to generate the electrospray and ensure that the frequency of the needle is three hertz.
Wait for 30 seconds for the measurement to be completed. After measuring each sample, dispose of the cartridge and needle in a biohazard disposer. To analyze the mass spectra, open the software associated with the mass spectrometer.
Click on the L-C-D file in the data file browser window of the software. Choosing click on a single peak of the mass spectrum window, and to automatically depict an E-I-C. Check the T-I-C and E-I-C, and click on the average spectrum icon to select the range of time window for generating the mass spectrum.
Export the mass spectral text data. To analyze body fluids, draw up to 10 microliters of the serum sample, and dispense it into a 1.5 milliliter microtube. Add 190 microliters of 50%ethanol to the tube, then vortex for two minutes at room temperature.
To eliminate all red blood cells, centrifuge the liquid at 15, 000 times G for one to five minutes at four degrees Celsius. Transfer 10 microliters of the supernatant into the well of the cartridge and proceed to measure on the mass spectrometer as previously. This protocol shows sample preparation for PESI-MS on solid sample, liquid sample, and living animal.
Human renal cell carcinoma shows characteristic peaks of neutral lipids. There were relatively strong peaks in the spectra at the mass-to-charge ratio of 900 from human renal cell carcinoma tissue that were not identified in the surrounding non-cancerous tissue. These peaks chiefly represent triacyl glycerol.
Example of data acquired in normal liver tissues, depicted the neoplastic lesion from a patient with chronic hepatitis and liver cirrhosis. Human serum from three individuals was analyzed using PESI-MS, there were clear differences in the spectral patterns among the individuals. For the metabolic changes in 5-FdU injected intraveneously into the tail vessel of a mouse, very rapid and sensitive detection of 5-FdU with sodium adduct was achieved in the liver in situ.
As the depth of the needle tip from the site of the tissue, plays the most important role in ionization that reads the successful data acquisition, make sure to follow the instructions. You can identify the molecular species by looking into the commercial database. This is a way to qualify the molecular mechanism of disease.
Many researchers have become interested in the application of this technique for predicting the prosperity of disease and it's prognosis. Especially using Ricket biopsy. Just make sure not to be pricked by the probe needle.
This can be simply avoided with a specific instrument for removing the needle from the machine.
