REACTIVE SPECIES - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 14

Protein/ligand
complex

Protein

Expose to synchrotron
X-rays

Creation of stable
oxidation products

Digestion with specific
proteases

Generate unique
peptide fragments

Compare oxidation rates for the
protein and the protein/ligand
complex

Identify oxidation sites by
tandem MS

50k

100

DAFLEKLPEN

y6

40k

b7
y7

30k

y3

y5

90

20k

In the absence of a ligand
In the presence of a ligand

b3 b4

b9

10k

y4

b5 b6

y8

y2 b8

0

80

0 50 100 150 200 200 400 600 800 1000

X-Ray exposure time (ms) m / z

Figure 1.5. Schematic representation of hydroxyl radical footprinting (adapted from
Xu and Chance [87] with the permission of the American Chemical Society).

conformations. Protein cleavage can be performed either enzymatically or
chemically for MS analysis. Common proteases used are trypsin, chymotrypsin,
pepsin, savinase, proteinase K, endoproteinase Asp-N, endoproteinase Lys-C,
and metalloendopeptidase Lys-N [90, 92, 146]. A mixture of several proteases
has been used to achieve cleavage at a wide range of sites in proteins [147].
However, protease is large and may not have access to the structural backbone
of protein for cleavage in order to probe the structure precisely. A microwave
method has also been applied to protein digestion [148]. Cyanogen bromide