Hello everyone,
My question is the next : Why the DHB matrix is a reducing agent while sDHB matrix is not a reducing agent?
In this article it is written that sDHB is a non reducing agent
Article Fast QC of intact monoclonal antibodies using MALDI-top-down...
In this review it is written that DHB is a reducing agent
Article Principles of hydrogen radical mediated peptide/protein frag...
I am working with ISD (Top down sequencing) on a nanobody (VHH) thats contains 2 cysteins. My signal with sDHB is weak and I can only match around 10 aminoacids for the full peptidic sequence. I have not tried yet with the DHB. Do you think that it can help me?
Also I would like to perform T3 sequencing (Pseudo MS3)on my system in the near future. Can I work with DHB for T3 sequencing or should I use the DAN matrix?
Also If you have tricks to perform ISD and it will be nice.
Best Regards,
Sébastien
Hi Sébastien
Have a look to the following Tanaka´s group work
Article Rapid sequencing and disulfide mapping of peptides containin...
They show that intermolecular disulfide bonds of the two chains of bovine insulin are reduced by 1,5-DAN but not by DHB. Since, as you may know, sDHB is a 9:1 mixture of DHB and 2-hydroxy-5-methoxybenzoic acid, I would try other matrices.
Another group has published an interesting work regarding this
Article Rational Selection of the Optimum MALDI Matrix for Top-Down ...
They have got nice results using a mixture of 1,5-DAN and picolinic acid as additive.
I would also check some minor things, such if your MALDI-TOF laser is properly tuned for ISD and the quality of your sDHB matrix. I have used the one from Sigma and have got nice results.
Hope this can help you, good luck.
Hi Newton,
Thank you for your nice answer. I use the same sDHB from Sigma.
More I read about ISD and N and C terminal sequencing and more I think that the DAN is better than the sDHB.
Do you work with 1,5-DAN matrix?
Also have you tried the T3 sequencing to get the amino acids from the beginning of the N and C terminii?
Best,
Sébastien
Hi Sébastien,
I heard elsewhere that DAN should be more effective to read N-terminal sequences and sDHB would be better to C-terminal, but I don´t know if it is always true. I think this has to be tested and will depend on the analyte you are studying.
Yes, I have used 1,5-DAN from Sigma and have got nice results to N-terminal sequencing of recombinant proteins expressed in bacteria.
I have not tried T3 sequencing yet but it seems to be quiet interesting to cover the whole protein sequence.
Regards
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