The overall goal of this procedure is to analyze intact proteins larger than 100 kilodaltons in a sensitive manner by maldi mass spectrometry. This is accomplished by first preparing the matrix solutions. The second step is to deposit the thin layer solution on the target.
Next, the sample and the mix of matrices are deposited on the target. The final step is to observe the sample spots under the microscope. Ultimately, a multi to mass spectrometer is used to analyze intact proteins and record mass spectra.
The main advantage of maldi mass spectrometry over other analytical approaches, such as electro spray ionization, is that it is more tolerant to salt, contaminant and detergents. Another advantage of maldi is that it generates ion carrying fewer charges compared to electro spray. Therefore, acquiring and interpreting MDI data is quite straightforward.
To begin the rech crystallization, pour 10 milliliters of 40%ethanol in a Pyrex que. Then at 600 milligrams of a matrix using a water bath and a resistance heater. Warm the matrix solution and stir it until the matrix is completely dissolved.
Repeat the previous steps until the solution is saturated. Allow the solution to cool slowly at room temperature for several hours, and then store at four degrees Celsius overnight. If crystals are not formed, scratch the inside of the beaker just below the solution surface, using a glass rod to induce crystallization.
Finally, collect the matrix crystals by filtration. Rinse the residual crystals in the flask with a minimum amount of ice cold solvent and filtrate them. Once filtration is complete, allow the crystals to dry to clean the maldi target plate.
Rinse it with methanol and wipe it gently with a kim wipe. Then rinse it with water and wipe it with a kim wipe. Insert the maldi target in a 600 milliliter beaker and cover it with 50%ethanol.
Next, sonicate the target in the ethanol solution for 10 minutes in an ultrasonic bath. If there are residues remaining on the plate, repeat the previous steps. Rinse the target with methanol or water the solvent used, determines how much the sample spreads.
Then tilt the target so that all of the liquid is collected on a Kim wipe. Dry the target using a nitrogen gas flow. In the next step, dissolve alpha CHCA in acetone.
In order to make a saturated solution, dip a 10 microliter pipette tip in the alpha CHCA saturated solution so that a small amount of the solution flows in the tip. Touch the maldi target rapidly with the pipette tip to deposit the alpha CHCA solution on the maldi target using a 1.5 milliliter plastic tube. Prepare a 20 milligram per milliliter alpha CHCA solution by dissolving alpha CHCA in a seven to three volume ratio of Acetonitrile to 5%formic acid, labeled the solution as alpha CHCA solution.
Following this, prepare a 20 milligram per milliliter DHB solution by dissolving DHB in a seven to three volume ratio of acetyl nitrile to 0.1%Tri fluoro acetic acid, labeled the solution as DHB solution. Mix the alpha CHCA solution and the DHB solution in a one-to-one volume ratio. To obtain the C-H-C-A-D-H-B solution before starting this procedure, an optional step is to purify the protein samples by buffer exchange shown in the listed reference.
Otherwise, deposit an un purified protein sample on the MALDI target. Next, deposit 0.5 microliters of the protein sample on the prepared alpha CHCA thin layer. Immediately after this, add 0.5 microliters of the C-H-C-A-D-H-B solution.
Deposit 0.5 microliters of the Cain standard on the MALDI plate, and then add 0.5 microliters of the matrix solution. Once the samples and the cain are dried, you can observe the spots under a microscope, insert the target into the instrument, and choose the appropriate instrument setup. Then choose the appropriate mass to charge ratio range.
Acquire the spectra of the ca and calibrate the instrument using the appropriate laser intensity. Acquire the spectra of the protein samples. MALDI toff analysis of the intact chromosome region Maintenance one protein is shown here.
The C-H-C-A-D-H-B mixture yielded mass spectra of higher quality in terms of signal to noise ratio and sensitivity. In particular, 0.5 picomoles of protein deposited on the MALDI target could be detected, whereas the same amount of protein was barely detectable Using SINO NIC acid utilizing the C-H-C-A-D-H-B mixture, multiply charged ions of the protein were observed, allowing the mass determination with higher accuracy because the peak resolution is inversely proportional to the master charge ratio in the case of an axial multi toff instrument. Moreover, using the C-H-C-A-D-H-B mixture, our method of choice for the matrix deposition was the thin layer approach.
Using the dried droplet method proteins with a mass higher than 100 kilodaltons were not detected. The monomeric beta collect aase protein was, was analyzed by multi TOF as well. The C-H-C-A-D-H-B spectra were better than the SINO NIC acid spectra in terms of sensitivity and resolution.
Furthermore, the presence of multiply charged ions enabled confirmation of the mass of the protein with higher accuracy. MALDI to analysis of an intact immunoglobulin is shown here. The spectra obtained using the C-H-C-A-D-H-B mixture were much more intense than the SA and alpha CHCA spectra.
More specifically, the protein signals were higher and had better resolution in the C-H-C-A-D-H-B spectra. The use of the C-H-C-A-D-H-B mixture and the thin layer deposition method demonstrated significant improvements in the quality of large intact protein. Mass Spectra acquired using a MALDI to instrument.
The presented protocol illustrates how to acquire high quality mass spectra of high molecular weight proteins using a multi of instrument. While you're carrying out this type of analysis, three things are important. First, to use a freshly prepared metrics solution.
Second, to properly deposit your sample on the Mali target. And third, to use the appropriate instrumental setting such as laser intensity.
