I have three DNA samples about 5.5, 9.5 and 16.5 KDa, respectively. Now, I want to detect them by MALDI-MS.
Did anyone know which matrix is better for DNA detecting, 3-HPA, 2,4,6-THAP or 3,4-DABP? Is there any tips I should notice?
Thanks
Can you give us some more information about your project? For example, do you want to detect DNA itself (probably impossible), or DNA oligomers (sometimes diffiucult, but can be done depending on the number of bases)?
I would just spot a matrix of 3 samples vs 3 matrices, and try out. Desalting will be as critical as choice of matrix: you can try desalting your oligos e.g. on C18 ZipTips - try desalting with 50 mM NH4OAC solution instead of e.g. formic acid - or by using NH4-loaded Dowex cation exchange beads. Look to the literature - there are a lot of protocols out there for successful oligonucleotide analysis, e.g. Ross P et al. Nature Biotechnol. (1998). A lot of these protocols were worked out for SNP genotyping by MALDI-TOF in the late 90s. Finally, use linear mode. Positive and negative mode should both work; depending on the make and model of your MALDI find a source tuning that uses relatively long delay times, to make the extraction soft enough.
thanks you.
I used Amicon ultra 3K device for buffer changing and the DNA samples were finally dissolved in water, so there is no salt.
There's no matrix available in my lab now. I need buy one and can't decide which. Is there any difference between 3-HPA and 2,4,6-THAP?
I would probably vote for 3-HPA, a conservative choice that also works for larger >5 kDa oligos. THAP works better for shorter oligos, the third matrix I do not know. Re the use of amicons - try and see. They will help remove excess salt, but not necessarily the Na and K already bound, which usually is a lot. Your first spectra will show.
I used DHB and HCCA, which usually being used for petides detection. No doubt that no result.
Thanks for your help. I will try 3-HPA.
What's the concentration of your DNA?
We've some success in analzing synthetic oligonucleotides using 3-HPA.
We mixed 3HPA with diammonium citrate in 8:1 ratio.
First spotted and air-dried the matrix. Then, overlay the sample on top of the dried matrix for the analysis.
Typically, it requires at least 2 to 5pmol of the oligonucleotide
The concentration of DNA is about 2 μM.
what's the concentration of 3-HPA? 10 mg/ml? disolved in 50% ACE/ 50% H2O（V/V）？
Dear Kay
10 mg/ml 3-HPA dissolved in 50%ACN/H2O (v./v.), 10mg/ml DAC in H2O
Did you mean the final concentration?
will the ratio of 3-HPA with DAC affect the results?
My bad, ...I should have been more specific:
Please weigh 0.01g 3HPA and dissolve in 200ul of 50%ACN; in another tube, weigh 0.01g DAC and dissolve in 200ul of H2O
--> then mix 3HPA to DAC in 8:1 in a new tube for the final working matrix.
We did not further optimize the ratio as it seemed to work quite well.
Cheers
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA