In order to see the binding of a drug to proteins of interest, I used DARTS assay. In the 1D gel, one lane was loaded with crude protein sample and the other lane was loaded with crude protein drug mixture. Both the samples were treated with Pronase before loading into the wells. The image of the bands obtained after running the gel is attached. The lane on the left side is control (only protein). The two lanes (containing multiple proteins) were cut and sent for mass spectrometry analysis. m/z and intensity values obtained were analysed using peptide mass fingerprinting option in the Mascot online software. But I can't find my proteins of interest in the result. Am I not get an expected result due to presence of more than once protein in the lane?
Dear Charles sir,
First of all I would like to admit that only the protein that have been stabilized by drug binding need to be cut, not whole lane. You are getting multiple protein because of that.
The another solution for it may be as follows,
As single band in 1D gel is the mixture of proteins therefore in Maldi-tof you are getting multiple proteins. In order to get the specific protein, you need to run the protein in 2D gel for better results.
Hello Charles,
why have you used alpha-cyano 4-hydroxycinamic acid to ionize your control and DHB matrix to ionize your test sample?
Have you done in-gel digestion (trypsin or other enzyme) of your proteins before MS?
If not, then you should revise your MALDI-TOF analysis, looking for m/z ions higher than 5 KDa, using linear mode.
Conversely, your 1D gel result suggests some putative bands, at least 4, in the middle to up mass region. As Karan answered above, you may cut these bands individually, proceed trypsin digestion, then perform MS and MASCOT analysis. But, according to your result, I don´t think you need to run 2D gel.
Dear Charles,
How many gel sections/slices were made to carry out the in-gel tryptic digests? In the "MS Result", I only see spectra for one control and one test.
It will help to know details of the processing of the gel lanes.
Best,
Hediye.
Dear Charles, if you have access to an electrospray mass spec that might help: it seems that there are many adduct, SDS and salt species in that spectrum that probably confuse the analysis. Karan above is right about the gel bands and also make sure that your digestion is performed at the right conditions to make sure you have good enzymatic cleavage to make it as easy as possible to make the identifications from the bottom-up peptides. Also, you are welcome to a free trial of our search engine, Byonic, which specializes in protein identification, available at "proteinmetrics.com". The aspect that might help you is that we specialize if=n working out what is modified in the protein, so you should also be able to localise the drug.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides