protein mass spec problem

[Marcus, Dr J.]

Hi John, 

Your added 129 Da could be explained by an added glutamic 
acid, 125 Da would be explained by an additional glutimine 
or lysine residue, 105 comes close to the mass expected for 
an added cys (i.e. 103.2). How you are getting these 
additional residues from an expressed sequence is another 
matter which escapes me.  Is it possible that stop codon 
(TAA) could be mistaken for a glu codon (GAA)?  This might 
explain how you are getting the extra 129 Da.  This seems a 
bit far out but maybe it is worth considering.  Hope this 
helps.

Regards,
John Marcus

 
> Analysis of the pure protein by electrospray mass spec. produces four peaks.
> Peak one corresponds to the mass predicted from the gene sequence (40287).  
> Peak two is the mass of peak one plus 129.  
> Peak three is about 125 mass units larger than peak two.  
> Peak four is about 105 mass units larger still.  
> The heights of peaks one and two are similar, peak three is about half 
> as big, and peak four maybe half as much again.
> 
> Does anyone know why we might be getting these multiple forms?  
> 
> The protein was purified in Tris buffer, 
> 5mM ammonium sulphate, 1mM dithiothrietol and dialysed 
> against water before mass spec.
> 
> John Fox






_________________________________________________________
John Marcus            Marcus at tpp.uq.edu.au (Dr J.Marcus)
Cooperative Research Centre for Tropical Plant Pathology
5th Level John Hines Building
University of Queensland
St. Lucia, QLD 4072
AUSTRALIA

Fax: 61-7-3365-4771
Phone: 61-7-3365-4764