Protein Protocols Protein Protocols

684 Wada
Fig. 1. Detection of a variant protein by MALDI-TOF-MS. The mass separation is determined
to be 14 Da.
clearly differentiated in the mass spectrum taken at a resolution of 3000, and the mass
separation was determined from this analysis to be 14 Da (see Note 1).
The smallest difference in molecular masses detectable by MS taken at a modest
resolution of 2000–5000 increases with the increasing size of the proteins. A theoretical
analysis can be considered using two model proteins: β-globin (M r 15,867) and
transferrin (M r 79,556) (Fig. 2). A separation of 14 Da in transferrin is not clear (C in
Fig. 2B), whereas the shapes of the clusters for 20 and 31 Da differences, corresponding
to D and E, respectively, in Fig. 2B, are similar to those for 9 and 14 Da differences,
corresponding to C and D, respectively, of the β-globin in Fig. 2A. The minimum
difference in molecular mass detectable as pattern D in Fig. 2B for an equimolar
mixture with normal proteins of M r > 100,000 is about 20 Da. This means that about
half of the possible amino acid substitutions in a protein of this size will escape
detection. In those cases, a strategy to detect these substitutions preferably starts with
the analysis of CNBr-cleaved peptides (see Subheading 3.3.).
3.3. Chemical or Enzymatic Cleavage
3.3.1. Cleavage with CNBr Followed by MS
CNBr cleaves only at a relatively rare amino acid, methionine (Met), in proteins.
Therefore, it will generate large peptide fragments compared with enzymatic cleavage,
but their molecular size will be