6
Modifications were then added to the sequences. Because
insulin is comprised of multiple disulfide bonds linking the
alpha and beta chain, mass shifts were added to determine
the correct chemical formula (Figure 5).
Figure 4. The targeted insulin variant list was created by opening the “Add Protein” window (1), selecting means of importing variant
sequences (2), defining each entry as a peptide, and then selecting analytes to be included (4).
Figure 5. Mass shifts can be applied globally or locally by choosing Add/Edit Modifications (1). which opens the
working window (2). Here the specific amino acid residue to which the modification was applied was selected
and desired modification chosen (3).
For each insulin variant, three different disulfide bonds
were selected and assigned to three different Cys residues
as well as the addition of H
2
O to account for two chains.
Since the targeted quantitation was performed on the MS
signal, the specific Cys residues were not important.
After the sequences had the correct modifications
(chemical formulas), the
m/z
values were assigned
(Figure 6). Determination of precursor charge states and
isotopes linked the
m/z
values used for targeted data
extraction to the appropriate insulin variant sequence.