I am using a BIAM (N-(Biotinoyl)-N'-(Iodoacetyl)Ethylenediamine) probe to label reactive cysteine residues. I target to identify the labeling sites by LC MS/MS analysis. But the challenging part is I cannot identify the labeling sites yet, I have no clue what might have went wrong during LC MS/MS analysis. It would be really helpful if someone using a BIAM probe for similar research has identified the labeling sites and could help me in this regard.
I dont know if this will be helpful or not, but the iodoacetate group can react with a bunch of amino acids if you do the reaction at pH > 7.5 and using > 10-fold molar excess.
10 fold molar exess worked fine for me as well. You can as well add smaller amounts of TCEP to make sure your exposed cysteines are available as thiols.
How do you prepare your samples for MS analysis? We had quite some trouble with big fragments, that would not be detectable from extraction out of SDS gels but could be detected in precipitated samples.
Perhaps try another protein under the same conditions to find out if you have a problem with your conditions or your protein.
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