protein mass spec problem

John E. Fox altabios at
Tue May 6 10:02:56 EST 1997

Electrospray Mass Spectrometry on a purified recombinant protein.
Why multiple peaks?

We have expressed a recombinant subunit of a protein 
from a photosynthetic bacterium in E.coli.  
The subunit is soluble and easily purified.
It readily binds its substrate, indicating that it is properly folded.
It looks good on SDS-PAGE.  
N-terminal amino acid analysis of the subunit gives a 
reasonably clear sequence, identical to that predicted 
from the gene sequence.  

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

Alta BioScience                         Email: altabios at
School of Biochemistry                  Phone: 0121-414-5450
The University of Birmingham            Fax:   0121-414-3376
Edgbaston, BIRMINGHAM, B15 2TT, UK

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