We're trying to improve our sensitivity analysing proteins with our 5800 MALDI TOF/TOF. One thing that has come up is the purity of the sinapinic acid. Does it have an effect? We are buying it from Sigma and suspect we need to recrystallise it (as done for CHCA) but we are struggling to find a reliable method for doing so. If anyone can supply one or point me in the right direction it would be greatly appreciated.
Based upon intuition and the sensitivity of MALDI-TOF-MS (not to mention TOF-TOF) I would say that impurities could behave as ionization inhibitors or possibly energy sinks/insulators. I work with linear TOF, but I have a firm understanding of just how sensitive the technology is. What mass range are you working with?
As for re-crystalizing your matrix..... According to Sigma-Aldrich, this matrix is soluble in polar solvents. Maybe a column with a polar stationary phase and a series of solvents of increasing polarity?
At present we are optimising for MS Imaging, starting out with a mass range up to ~25kDa but in the future we want to go much bigger. As big as we practically can. So we're working in linear mode. If I didn't have to use the reflector for peptide work (and had some time) I'd love to play around with tuning the reflector for larger proteins.
The quality of matrix solution is very important for crystalization of samples. Additionally, CHCA is more suitable in case of analysis for large size of proteins.
I agree with Young-Su. CHCA can be purchased in bulk and cleaned and recrystalized quite easily. Sigma sells an impure CHCA for about $100 50mg (if I remember correctly). it is a bit yellow and therefore needs cleaned up and aliquots can then be made.
Good Luck,
Chris
We tested several different matrix supplier and finally decided to get all our matrices from proteochem (http://www.proteochem.com). They offer a wide range of matrices with a really high purity and no need for recrystalization. And: the matrix is relatively cheap...
I would also recommend that high quality test tube and pipette tip shall be used for sample preparation, Eppendorf works well. You can also use Eppendorf Protein LoBind tubes in order not to lose sample adsorbed onto the tube walls.
Keep in mind that also the solvents used for the solution have to be high grade to prevent impurities. Sometimes even MS-grade solvent charges can differ.
I used to recrystallise CHCA, and while it is time-consuming, it makes a massive difference when using a low-grade matrix.
If you use sublimation for applying the matrix, you can use much lower purity matrix. We use an imLayer and we find that the cheap CHCA is recrystallised as part of the sublimation process.
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