of nitration in mononitrated lysozyme is 26, 14, and 9 for ECD,
IRMPD, and CID, respectively. The number of fragments contain-
ing the sites of nitration in bis-nitrated lysozyme is 23, 13, and 8
for ECD, IRMPD, and CID, respectively. Thus, ECD produced
the largest number of nTyr-containing fragments in both cases.
ECD cleaved the protein backbone two and three positions
C-terminal of Tyr23 in mono- and bis-nitrated lysozyme, respec-
tively. However no ECD cleavages closer than eight positions
N-terminal of Tyr23 were produced. In contrast to that, IRMPD
cleaves much closer to the nitration site(s) and, furthermore,
cleaves between Tyr20 and Tyr23 in bis-nitrated lysozyme, Figure
2c. CID of the nitrated lysozyme is the least efficient of the three
MS/MS techniques, as discussed below.
Nitrated Cytochrome c. Unmodified cytochrome c ions with
a charge state z g 9+ produce fragments extensively under ECD
in our instrument with the exception of the region around the
covalent heme group attachment to the protein backbone.40
Previously we also pointed out the importance of searching for
c' and z ion fragments in ECD spectra of cytochrome c, as
for some charge states of cytochrome c their number exceeds
that of c' and z∙ fragments.40
Nitration of cytochrome c was not as efficient as in the cases
of myoglobin and lysozyme. Electrospray mass spectra of the
nitrated cytochrome c samples were dominated by the unmodified
protein ions with small peaks corresponding to mono- and bis-
nitrated protein (~20 and 15% of the abundance of the native
protein ions, respectively). LC separation of the sample containing
nitrated cytochrome c using a strong cation exchange column was
not successful. Thus a small isolation window of 10 Th was used
in order to isolate the ions of mononitrated protein from the ions
of both unmodified and bis-nitrated protein for MS/MS investiga-
tion. Such a narrow isolation led to a reduction in the number of
trapped ions and adversely affected the MS/MS analysis, i.e., the
majority of ECD fragments from the mononitrated protein were
not observed with intensities detectable above the noise level. ECD
of bis-nitrated cytochrome c was more efficient, as a larger
isolation m/z width could be used. Additionally, in all the MS/
MS experiments on cytochrome cthe most abundant charge state,
14+, was used in order to maximize the number of fragments.
MS/MS spectra and summaries of fragments from nitrated
cytochrome c are given in Figure 3. Although several ECD
fragments, including c, and z'-type ions, containing the possible
sites of nitration were found for bis-nitrated cytochrome c, the
total sequence coverage was still poor in comparison with
myoglobin and reduced lysozyme ECD data. CID of mono- and
bis-nitrated cytochrome c was not efficient and produced only
a few fragments at N-terminus (data not shown). IRMPD of
mononitrated cytochrome c was more efficient than ECD and
produced several backbone cleavages around the possible sites
of nitration, Figure 3b. Both the ECD and IRMPD data allowed
localization of the site(s) of nitration in the protein, and a
comparison could be made with other methods of nitration.
Cytochrome c has four tyrosine residues: Tyr48, Tyr67, Tyr74,
and Tyr97. Tyr74 and Tyr97 are solvent-exposed, and the mono-
nitration was expected to take place at either of them, as previously
observed with peroxynitrite as a nitrating agent.45,46 Tyr48 and
Tyr67 are less exposed, but Tyr67 is located close to Tyr 74 and
can be a target for secondary nitration. Batthyany et al.48 observed
nitration in bis-nitrated cytochrome c at Tyr74 and Tyr67 or Tyr97
and Tyr67. Interestingly, the authors did not report simultaneous
nitration at Tyr74 and Tyr97.
In our MS/MS experiments, IRMPD produced six and four
C-terminal fragments between Tyr74 and Tyr97 in mono- and bis-
nitrated protein, respectively, which did not contain the nitro
group. Four fragments, again without the nitro group, were
produced from the same region by ECD of bis-nitrated cytochrome
c. Only one C-terminal IRMPD fragment containing nTyr97 was
observed from mononitrated protein ((y294+)* in Figure 3b).
Furthermore, no fragments containing nTyr48 were observed for
ECD or IRMPD, and three unmodified N-terminal ECD fragments
between Tyr48 and Tyr67 indicated the absence of nitration at
Tyr48. Thus, while there is some evidence for initial nitration of
Tyr97, the majority of the data suggests that initial nitration occurs
at Tyr74. As Tyr48 and Tyr97 appear to be less probable sites of
nitration, the site of the secondary nitration is suggested to be
Tyr67.
Recently we reported abundant losses of neutral species,
including hydroxyl radicals, water, and ammonia, from nitrated
peptides following ECD.38 We have also recorded abundant losses
of neutral species from ECD of the intact nitrated proteins.
However, significant loss of neutrals even from the unmodified
proteins was present under ECD, as in the case of lysozyme,
Figure 4a. A significant increase in the relative abundances of the
fragment ions resulting from neutral losses was observed upon
nitration, Figure 4b, but the neutral losses due to the nitration
could not be easily separated from the original neutral losses from
the unmodified protein.
DISCUSSION
ECD was not completely inhibited in the vicinity of the site(s)
of nitration: it produced cleavages at a distance of two and three
residues from nTyr23 in mono- and bis-nitrated lysozyme, respec-
tively, Figure 2. However, the total number of cleavages in the
vicinity of the nitration site(s) is larger in the case of IRMPD for
all three proteins investigated. Table 2 summarizes the numbers
of cleavages produced by ECD or IRMPD within six residues N-
or C-terminal to the site(s) of nitration. IRMPD not only produced
more cleavages in that important region but also cleaved between
the two nitro-tyrosines in bis-nitrated lysozyme and cytochrome
c, while ECD did not, Figures 2 and 3. That result is consistent
with the previous reports on suppression of ECD by nitration in
doubly charged peptides and higher efficiency of the “slow-
heating” MS/MS methods in such cases.38,39 However, in contrast
to MS/MS of peptides, ECD provided the largest total number of
fragments from intact mononitrated myoglobin, mono- and bis-
nitrated reduced lysozyme, and bis-nitrated cytochrome c (includ-
ing c, and z' fragments).
Sequence coverage by ECD was greater for nitrated myoglobin
and reduced nitrated lysozyme than for nonreduced nitrated
lysozyme and nitrated cytochrome c, where other internal
modifications were present: disulfide bonds (lysozyme) and a
heme group (cytochrome c). In the case of ECD of nonreduced
nitrated lysozyme, the poor sequence coverage was due to the
(48) Batthyany, C.; Souza, J. M.; Duran, R.; Cassina, A.; Cervenansky, C.; Radi,
R. Biochemistry 2005, 44, 8038-8046.
Analytical Chemistry, Vol. 82, No. 17, September 1, 2010 7289