Dear colleagues,
We are trying to accurately determine the mass of a protein (e.g. 50KDa) by MALDI-TOF.
The protein has a concentration of approx. 3 pmol/ul, kept in 30 mM PBS buffer (no other MS-incompatible ingredients). It always produces a broad, poorly-resolved, low S/N peak in MALDI-TOF. The situation does not improve even after we've performed Ziptip desalting.
Would you please enlighten me on how to produce a sharp, stronger MS peak (for the protein sample)? May I also inquire:
- The standard / calibrant we used always produce a sharp and strong peak, but its concentration is also 3-4 pmol/ul
- Various articles suggest MALDI to be able to tolerate 50 mM PBS or less
- Even ziptip has no improvements
Retrospectively, is the issue (i.e. the ability to produce a sharp and strong MALDI-TOF peak, for a given protein), is mainly related to the individual sample’s physical/chemical properties?
Thank you very much!
Did you ever tried to do sandwich preparation? It is a tip Chrys Wesdemiotis gave me recently as he was at our institute for a talk. He said, his researchers always had improved S/N ratios when doing that preparation. I also had good results with that (as least, signal did not got worse).
How to do: apply 1µl matrix solution on target plate, let dry. Mix sample with matrix solution (1:1 or how you usually do that), apply 1µl on dried matrix spot, let dry. Apply another 1µl of matrix solution on top, let dry.
You will always get an envelope of masses for something as large as a protein, because you have a distribution of carbon 13 content. If you are not able to resolve 1Da differences in 50 kDa this will give a broad peak. You can also get various ionisation states (different charges), depending on the machine you are using. These two issues can combine to give a very broad spectrum. Additionally, depending on your protein, there may be post translational modifications, such as glycosylations, acylations etc, which may give a range of masses that you are not able to resolve.
Hi Kay,
Some proteins are surprisingly showing better signal if you increase ratio of the sinapic acid. I usually use ratio 1:2 (protein:matrix for dried droplet) as a starting point, but sometimes I've got better intensity with 1:3 or 1:4 ratio. Definitely, PBS is not recommended as a good buffer, I would recommend to try classic NH4HCO3, if possible. You can try BioSpin columns for changing buffers.
Which type of ZipTips have you used? C4 or C18? For proteins, C18 is too hydrophobic and your protein may remain bound in the tip.
Lukas
... and yes. Ionization potential is very specific for each protein and is dependent, besides other things, on the AA sequence, modifications, mood of the instrument :), etc. This is also one of the reasons, why peptides after digestion don't have the same signal intensity.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides