REACTIVE SPECIES - Oxidation of Amino Acids, Peptides, and Proteins: Kinetics and Mechanism - page 19

* [M + H] +

y

b

*

trypsi n

ESI CID

500 600 700 100 400 700

m/z m/z

A

MS MS/MS

B

* [M + 3H] 3+ [M + 3H] 2+

&[M + 2H] 2+

Lys-C/
Asp-N

z

*

ESI ETD

c

500 600 700

400 600 800

m/z

m/z

MS/MS

MS

C

[M + 3H] 3+ [M + 3H] 2+
&[M + 2H] 2+

z

ESI ECD

*

c

11,000 12,000

1000 1200 1400

m/z

m/z

MS MS/MS

Figure 1.7. MS-based approaches A, B, and C. The gray boxes symbolize what takes
place inside the MS equipment; the stars denote a certain peptide or protein (from an
oxidized sample), chosen for CID, ETD, or ECD (adapted from Törnvall [90] with the
permission of the Royal Society of Chemistry). See color insert.

more cleavages were generated in the vicinity of the sites of nitration in CID
than IRMPD and ECD. ELDI is suitable for MS and top-down MS/MS of
large proteins up to 29 kDa [164, 165]. The use of LC ESI tandem mass spec-
trometry (MS/MS) to sequence peptides generated from a digest of proteome
sample has also been demonstrated [158].
MS-based approaches are generalized in Figure 1.7 [90]. In the bottom-up
approach (A), tryptic proteolysis is followed by MS and CID-MS/MS analysis
of relatively small +1 and +2 charged species, while further species proteolysis
is followed by MS and ETD-MS/MS of larger +2, +3, and +4 charged species
in the middle-down approach. A top-down approach involves ESI-MS on the
intact protein to determine the total protein mass followed by ECD-MS/MS
of a selected protein species with the modification of interest. The bottom-up
approach is the most commonly used method for peptide mass analysis. Several
labeling and separation methods are applied in the analysis of complex protein
mixtures.