I would like to find out if there exists a chemical probe that can be targeted to a specific site on a protein, that has been genetically labeled without any unnatural amino acid incorporation, such that proteins in close proximity to that site can then be covalently labeled for mass spec identification. For example, if a chemical reagent has high affinity for a certain amino acid sequence, and this reagent can be photoactivated to expose a reactive group that then alkylates nearby protein residues, and this alkylation either installs an affinity tag for pulldowns or a label that can be detected directly on mass spec. Thanks!
If you have the capability of genetic engineering of your protein, then you can set it up with a single surface-exposed cysteine residue. Many reagents are available that react selectively with cysteine. A bifunctional reagent with a maleimide (for example) on one end and an azide (or other reactive group such as a succinimidyl ester) should satisfy your requirements. Here is one example with azide:
http://www.kerafast.com/p-1063-azide-peg3-maleimide-kit.aspx
Also check out bifunctional crosslinkers at Thermo-Fisher Scientific.
Thanks, Dr. Shapiro. I was hoping for something that would be more site-specific: many proteins in a complex mixture such as a lysate (and several sites on my protein, unless I mutate it) will have cysteines that will be equally likely to get crosslinked. I am not working with a single purified protein...
This review from the Schepartz lab at Yale discusses several techniques:
http://www.schepartzlab.yale.edu/publication/pdfs/Lowder2011.pdf
and the more recent paper from the Bonasio lab at NYU has an interesting method:
http://www.ncbi.nlm.nih.gov/pubmed/25311790
Tetracystein tagged from Roger Tsien lab is a classical option: use these key words on pubmed: tsien flash. In particular Nature Protocol 2010.
Another option that I know about is the dimaleimide coumarin reagents described by the Keillor lab. Use these keywords on pubmed: keillor maleimide . See also the link below.
Both wprk with tags containing multiple cysteins in a precise spacing ensuring better specificity than single cystein reagents.
Thank you, Francois-Xavier and Edward, those were useful suggestions. The tags that are specific for tags with multiple cysteines are better than alkylating single cysteines. I'm sure there might be some off-target reactions though - I will BLAST my organism's proteome to see what all might naturally contain the motif... If just one or two sites, I might be able to CRISPR/Cas it away!
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