There are many ways to tackle this but probably the simplest to start with would be to do a in gel digest (using trypsin) followed LC MSMS. The coverage will depend on a number of factors including protein source (well chacterized organism =comprehensive data base=better coverage), the protein sequence, type of MS you have available to do the analysis (high res /high mass accuracy instrument =better coverage). To improve the coverage can follow up the in gel digest with some digests using complementary enzymes to trypsin. 

Thank you for the information. The primary sequence of the protein is not known, but can I at least find the number of cysteine residues  present in the protein molecule by performing tryptic digestion followed by ESI-MS? 

Sure, but it depends on the factors mentioned above. You probably won't get 100 % sequence coverage but will be a good start.

What type of mass spec do you have available to do the analysis? Also, what is the source of your protein? 

Dear Pooja Nand,

You may not able to find the Number of Cys present in the Protein by ESI-MS because from ESI-MS you will get M, M+, M+2+ exactly more then this fragment unable to give clear information by ESI-MS

You have said the primary sequence not known so that you use LC-MS-MS means you can able to know the number of Cys details but it also may not be clear.,

You just get sample and go for amino acid sequence composition analysis from this you will get all amino acids presence and absence, if your compound is not much pure or unknown target with mixture also you can able to find number of Cys by using amino acid sequence/composition finder

Now latest amino acid sequence finder available it will give some approximate % and amino acid composition details so that you can able to get clear information.,

If You need to analyze or any technical problem to find amino acid composition ask me back,  

Hope you clear now!

Good Luck & Regards

R.Selva...   

The two techniques are complementary. Agree that amino acid analysis will give you the answer to number of Cys residues present whilst MS will give you the identity of the protein as well as Cys positions. So, really depends on the ultimate aim of your study. 

Dear Dr. Stoychev,

Thank you for the information. We have ESI-MS available at our institute and the enzyme (obtained from a bacterial source) was bought from Sigma Aldrich.  It is available in its powder form.

Dear Selva,

Thank you for the information.

There should be enough info on the Sigma website to obtain the sequence of the protein, presumably you have the name as well as species. You can than do a Unirprot search for the protein sequence (http://www.uniprot.org/). Sigma also typically provides the EC number which can than be searched using: http://www.brenda-enzymes.org/

Rgd your MS system, there are many different ESI-MS instruments. To get good sequence coverage better to use a high-resolution/ high mass accuracy instrument such as the new generation Orbitrap or QTOF systems.

Dear Dr Stoychev,

I have already gone through these Protein structure info sites. But the activity of the enzyme which I m using is different from the activity of the enzyme listed in these website. (though the both the enzymes are from the same microbe Bacillus fastidiosus) Probably, the organism may be of the same species and genes but may be a different strain. Hence the difference in activity.

The difference in activity also indicates a difference in the structure and distribution of cysteine residues. Hence I am interested in determining the structure of the enzyme bought by us commercially.

Depending on the size of your protein, you can easily determine the number of Cys in your protein using MS. First determine the mass of the native protein, reduce and alkylate then determine the mass again. Since you know the mass of the alkylating agent, simple maths will tell you how many Cys are present in your protein.

Using this technique you can also determine the number of disulphide bonds if required. In this case, determine the native mass, alkylate only (do not reduce first), and determine the mass. Then reduce and alkylate with a different agent and determine the mass. Again, simple maths will let you determine the number of Cys and how many disulphide bonds are present in your protein.

All this assumes you have the protein present in a sufficiently pure state (which you should), the protein is not too large to determine a relatively accurate mass in your MS and related to this the resolution of the MS is sufficient.

Dear Dr. Hains 

Thank you for the information.