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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.