multiple antigenic peptides - MALDI

Paul Morrison morrison at FARBER.HARVARD.EDU
Wed Jun 8 16:37:48 EST 1994



>In article <2t20v0$cl6 at sun4.bham.ac.uk>, altabios at bham.ac.uk (John E. Fox)
>writes:
>> We synthesise custom multiple antigenic peptides, (octomeric peptides)
>>according to 
>> the method of James Tamm, Immunol Methods (1989) 124, 53-61.
>> As part of our quality control checks, we have tried to analyse them on a
>>MALDI mass 
>> spec. using gentisic acid as the matrix. 
>> We see no sensible spectrum at all for any of the peptides we have made. 
>> These peptides have molecular wieghts roughly in the range of 10KDa to
>>18KDa, 
>> so they are well within the working range of our machine, (a Kratos MALDI 3).
>> We have tried them on another machine with exactly the same results.
>> 
>> Has anyone  else tried to mass spec these compounds? Anyone with an idea of
>>why
>> we have failed to see their spectra?
>
>John;
>        We also had trouble with this type of peptide (8K Tamm) but finally
>got reasonable 
>spectra from a dilute solution of matrix with concentrated peptide and low
>laser power.
>        Have you tried different matrices? Our preferences for peptides
>of this size are (in order of preference) 
>1) alpha cyano-4-hydroxy cinnamic acid 
>2) 3,5-methoxy-4-hydroxy-cinnamic acid 
>3) 2,5 dihydroxybenzoic acid. 
>        The spec we have (VG TofSpec) has slightly different capabilities
>vis-a-vis laser power and density than your MALDI III but I don't think these
>differences are the deciding factor.
>
>Hope this helps.
>
>Lorne
>
>ltaylor at peptidarus.uwaterloo.ca


We have found MAP peptides difficult to analyze by MALDI/TOF and impossible
to analyze by RP HPLC so we adulterate the MAP resin with the single amino
acid resin representing the C terminus. This way in MALD/TOF analysis  if a
full length single chain is represented our confidence that the MAP peptide
is OK goes up. In both RP/HPLC and MALD/TOF the MAP peptide is going to be
a "forest of peaks". On the down-side this single chain as full length is
going to tell me nothing about steric hinderance occuring in the growing
MAP. We also do amino acid analysis and if the ratio of lysine tree,
alanine core amino acid to amino acids in the chain look good then
confidence level again goes up.
I agree with Lorne on his matrix. We fly all of this size peptide on alpha
cyano-4-hydroxy cinnamic acid.
Hope this helps, Paul






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