Here's a protocol for how to do just that:

http://openwetware.org/wiki/Free_Sulfhydryl_Determination

Send a sample to the closest MS facility.

Here's a protocol for how to do just that:

http://openwetware.org/wiki/Free_Sulfhydryl_Determination

If the cysteine is exposed, you can conjugate it with a fluorescence probe such as I-AEDANS (obtained from Molecular Probes). The procedure would be fairly quick, and the presence of AEDANS conjugate would be obvious from the fluorescence signature.

In addition to the above answers, any of the titrations (NEM, pHMB or DTNB) can be used to determine free sulfhydryl group(s). The protocols are available in the original references of these methods.

Use Ellman's reagent, 5-5dithiobis[2nitrobenzoic acid] (DTNB). E.g. see http://www.filgen.jp/Product/Bioscience4/exocell/1020%20Universal%20TBARS%20Insert.pdf for a commercial kit, or for a protocol:

http://online.sfsu.edu/gassner/Chem%20343/DTNB%20Assay%20for%20Free%20Thiol.pdf. Here is Pierce's product sheet that has a protocol and the original reference: https://www.piercenet.com/instructions/2160311.pdf.

I recall a paper that is very germane to your your problem:

T.E. Creighton, Nature 284:487-489, 1980

One can count the number of available cysteines by introducing varying concentration ratios of iodoacetate and iodoacetamide with the protein solution, and then subjecting the protein to electrophoresis under conditions in which its mobility is determined only by its net charge.

good luck.

The classic method to use is easy, and takes about 1/2 hour. It uses ELLmans reagent. The protocol is on the internet. But basically, you take the reagent and dissolve in water. Then you add an aliquot to your protein. A yellow color forms from breakage of the disulfide bridge in the reagent to form the pyridinylthiolate ion. The absorbance at about 405nm tells you how many free cysteine there are. The extinction coefficient for the product is known. If you need something more sensitive, you can label the free cysteines with c14 iodoacetic acid.

For the c14 iodoacetic acid experiment, you add the acid and allow it to incubate. It would have a known specific activity so that you can calculate from the number of counts, how much label is present in terms of mole equivalents. After incubation, a spin column can be used ie like a zebra column ( desalting) to separate unreacted reagent from you protein. Then you just use a scintillation counter for the protein sample.

The Ellman’s protocol can be used to determine the concentration of cysteine residues in a purified protein. It is based on reaction of the sulfhydryl with DTND (5,5'-Dithiobis-(2-Nitrobenzoic Acid). DTNB + SH -> 2-nitro-5-thiobenzoic acid (yellow). A calibration curve is generated by reaction a sulfhydryl compound of known concentrations (cysteine, DTT, beta-ME and etc.). For examples, using serial dilution of DTT, run reaction with DTND for at least 5 min and measure absorbance at 412 nm. The extinction coefficient of the yellow compound at 412 nm is 13,600. Details you will find in those two publications. Their protocols could be modified. Sure that different methods to determine free cysteine in proteins could be found in publication or on Internet.

References

1) Ellman, G. L. (1959). Arch. Biochem. Biophys., 82, 70-77.

2) Bulaj, G., Kortemme, T., Goldenberg, D. P. (1998). Biochemistry, 37, 8965-8972

Incubate your protein with any of the cysteine reactive dye (DTNB, MMTS, I-AEDANS or even glutathion) at pH ~8. Under physiological conditions surface exposed cysteine will react with the dye in couple of minutes, which can be detected using either optical methods or by mass spectrometry. If cysteine is buried, you may add some denaturant during labeling. For detail you may refer to the link below. I hope this will help you.

Nilesh

http://www.ncbi.nlm.nih.gov/pubmed/8679642