Hi Safaa! Sorry I am a food technologist and hence do not know about your protein of interest. Do you know the number of cysteines in your protein sequence? Also the MS methodology is based on the ability of the protein to be charged. Thus, if the protein has a number of lysines and arginines or aspartic and glutamic acid residues in the sequence, it may be possible to get good signals.

Also do remember that most MS analysis protocols require disulfide bonds to be reduced and alkylated for easy identification. So if you are interested in the disulfide bonding between specific cysteines, other methods e.g. X-ray chrystallography may be more useful than MS. Hope this information was helpful!

Thank you so much Anant Dave and John S Wishnok! Actually, am new with both (MALDI and protein chemistry). My protein has 25-27 cysteines (no disulfide bond). I am interested in the activity of anticancer drugs towards keap1-Nrf2 pathway. Also, interested in how/what of the cysteines are modified. My major goal, is how to decrease the influence of keap1-Nrf2 pathway on the chemotherapy resistance in colon cancer. I have to do the MS experiments by myself. I can`t work with intact protein (65k) that`s out of the range of our MALDI-TOF. I will work with digested keap1 by trypsin.

Hello Safaa,

I would say you about the skills of your experiment it is important to consider which matrix you use and chemical parameters of matrix solution ( solvent, pH and concentration) to get better signals intensity. If you want to study the reactivity of cysteine in your peptide you could use compounds/anticancer drugs with different electrophilicity.  

Regards,

Marco

Hi Safaa,

a Protein with 25 to 27 free Cys without any disulfide is tricky to handle I guess. As soon as you dissolve it water or buffer in presence of oxygen or oxidating agents, you will get disulfide bonds at pH > 5.

To check the reactivity of the Cysteins (some of them may in the hydrophobic cehnter of the peptide and not facing to the outside) you could do a derivatization with a Maleimide component (e.g. Atto dye). Dissolve the protein under acidic conditions (at pH

An addition to what Marcus said:

your MS instrument may tell you also WHERE a modification happens if you can digest the protein.

If this is your goal, MS/MS would be a good addition to your analytical capabilities 

Sorry Safaa, I forgot the Sigma Aldrich link yesterday... there you go. Thanks to Fabrizio, he is right - digestion will help, but still it's a tough challange, I believe.

http://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=103431784

Marcus Binner, thank you so much for your suggestion. Really, they are very helpful and I work on. 

Fabrizio Donnarumma,Marco Giampà: Thank you for your answer. Yes! my goal to find out which cycsteines are modified.

Thanks again for ya`ll.