I need to develop a method to detect the mass of an oligonucleotide 10kDa using Microflex LT system Bruker Daltonik and Flex control sofware.
The programme has already some methods, such as LN 0-3kDa, but I did not manage to change the method to detect my molecule.
Dear Simona,
I would test first 3-HPA (3-hydroxypicolinic acid) matrix. You may use it saturated in 50% ACN, containing 10 mg/ml diammonium hydrogen citrate (DHC), applying sample by dried droplet method and analysing in linear negative mode.
You may also test other two matrices: THPA (2,4,6-Trihydroxyacetophenone) with triammonium citrate as additive and DHBP (3,4-diaminobenzophenone).
Have a look to this paper Article A matrix of 3,4-diaminobenzophenone for the analysis of olig...
Good luck
Thanks for your advice.
At the moment I am using 2,5-Dihydroxybenzoic acid DHB at a concentration of 5 g/L in 0.1% TFA solution containing 30% acetonitrile and 1 g/L of ammonium phosphate monobasic. 0.5 µL Matrix solution deposited onto the MALDI target and 0.5 µL analyte solution.
We managed to see a very low signal. I don't know if this is due to the matrix, the sample concentration or if there is something wrong in my parameter setting.
I am talking about parameters such as Ion source 1, Ion source 2, Lens etc...
Simona,
I´ve never used DHB to ionize oligonucleotides since it is not recommended to do that. It also looks strange the phosphate usage as additive, I´ve never heard about that too. Anyway, if you are using a good instrument I would not mind with ion source and lens voltages, indeed. I would not expect any improvement by changing ion source voltages in this case. Lens voltage will benefit your signal depending on m/z range you are analysing. If you are interested in 10 KDa range your voltages may be between 8 and 10 KV without significant changes in your signal. You can test it and see what I mean. Conversely, laser power would be more infomative to you. How much laser power you are using? I would care with sample quantities. If you are using low fentomol your signal will be probably weak, mainly if your sample keeps sodium salts. Consider change your matrix, as I recommended you before, and check your sample concentration and alkali content.
Hope it helps.
Look up the patents by Sequenom and Christopher Becker. Chris had a company that was doing this and Sequenom held the NIH contracts for re-sequencing the human genome by MS for many years. I think both companies are now out of buisness and the patents are now expired.
I agree with Newton. Have a look at the Bruker's Guide :https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/InstructionForUse/8702557_IFU_Bruker_Guide_MALDI_Sample_Preparation_Revision_E.pdf
Many thanks.
I will change matrix. I will try 3-HPA + diammonium hydrogen citrate
From Bruker manual:
"Prepare a half-concentrated solution of 3-HPA in TA50 solvent (50:50[v:v] acetonitrile : 0.1% TFA in water) containing 10mg/mL diammonium hydrogen citrate (C6H8O7 * 2NH3 )"
Hopefully I will manage to detect my oligo!
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