thank you to all those responding to my question about storage and
purification of alkylating reagents, this message is a supplementary
question on the same subject.
We are doing ESI-MS experiments on whole recombinant proteins to establish
disulphide bond formation. Our approach is to purify monomer away from
oligomer by gel filtration chromatography and then perform either
alkylation or reduction/alkylation and compare MW's by ESI-MS.
We have the situation where a protein with three cysteines increases in MW
by one alkylating group prior to reduction and by three alkylating groups
after reduction, exactly what we were anticipating, one free cysteine and
one disulphide.
The problem is that there is another smaller (perhaps 5% by peak height)
species in the spectra representing two alkylation sites prior to reduction
and four after reduction.
Does this mean that both the 4-vinyl-pyridine and the iodoacetamide are
alkylating some other reside besides cysteine or is there some other
explanation that has escaped me?
Regards....Ken
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Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.medstv.unimelb.edu.au/abrf.html
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