myoglobin fragment extensively under ECD in our instrument.40
ECD of unmodified cytochrome c does not produce fragments
from the vicinity of Cys14 and Cys17, where the heme group is
covalently attached to the protein.40,41 Native lysozyme has four
disulfide bonds, which have to be cleaved by ECD first in order
to produce backbone fragments from the interior of the molecule,
i.e., multiple electron capture is required. Thus nitrated myoglobin
represents a system where ECD can be affected only by the
presence of the nitrated tyrosine, while native lysozyme and
cytochrome c represent systems where ECD efficiency can be
affected by other modifications in addition to nitration. For
comparison, ECD, CID, and IRMPD of reduced and alkylated
nitrated lysozyme were also carried out. Finally, we compare our
results for top-down ECD and IRMPD of these nitrated proteins
with our recent results on ECD of peptides containing 3-nitroty-
rosine.38
EXPERIMENTAL SECTION
Materials. In this work we used methanol (Fisher Scientific,
Leicestershire, U.K.), water (J. T. Baker, Deventer, The
Netherlands), formic acid (Fisher Scientific), nitric acid, diso-
dium tetraborate, boric acid, sodium nitrite, sodium phosphate,
sodium chloride, ammonium acetate, and ammonium bicarbon-
ate (Fisher Scientific), dithiothreitol (DTT), and iodoacetamide
(IAA) (Sigma-Aldrich, Poole, Dorset, U.K.). Equine skeletal
myoglobin, chicken egg lysozyme, and cytochrome c (horse
heart) were purchased from Sigma-Aldrich and used without
further purification.
Preparation of Nitrated Proteins for MS/MS Analysis. Our
aim was to prepare nitro-proteins in the state(s) of nitration found
in vivo. Electrosynthetic modification of proteins has been shown,
under various conditions, to oxidize specific amino acid residues
including tyrosine.42-45 Although in vitro chemical nitration may
perhaps provide larger total yields of modified proteins, it has been
observed to be less selective and specific than electrochemical
nitration. For example, in vitro nitration of lysozyme and cyto-
chrome c using peroxynitrite46,47 results in combinations of mono-,
bis-, and tris-nitration, among other PTM modifications, and
complex separation of products is necessary. Furthermore,
analysis of tryptic hydrolysates of lysozyme nitrated in vitro by
myeloperoxidase revealed that Tyr23/Trp28 were modified to a
higher extent than Tyr20 (specific site of nitration) and additionally
that Trp62/Trp63 were oxidized and nitrated.46 The same residues
were also the main targets of peroxynitrite, which also hydroxy-
lated Trp108/Trp111.
(40) Mikhailov, V. A.; Cooper, H. J. J. Am. Soc. Mass Spectrom. 2009,20, 763-
771.
(41) Horn, D. M.; Breuker, K.; Frank, A. J.; McLafferty, F. W. J. Am. Chem.
Soc. 2001, 123, 9792-9799.
(42) Kendall, G.; Cooper, H. J.; Heptinstall, J.; Derrick, P. J.; Walton, D. J.;
Peterson, I. R. Arch. Biochem. Biophyy. 2001, 392, 169-179.
(43) Matters, D.; Cooper, H. J.; McDonnell, L.; Iniesta, J.; Heptinstall, J.; Derrick,
P.; Walton, D.; Peterson, I. Anal. Biochem.. 2006, 356, 171-181.
(44) Iniesta, J.; Esclapez-Vicente, M. D.; Heptinstall, J.; Walton, D. J.; Peterson,
I. R.; Mikhailov, V. A.; Cooper, H. J. Enzyme Microbi Technol. 2010, 46,
472-478.
(45) Iniesta, J.; Cooper, H. J.; Marshall, A. G.; Heptinstall, J.; Walton, D. J.;
Peterson, I. R. Arch. Biochem. Biophys. 2008, 474, 1-7.
(46) Vaz, S. M.; Prado, F. M.; Di Mascio, P.; Augusto, O. Arch. Biochem. Biophys.
2009, 484, 127-133.
(47) Abriata, L. A.; Cassina, A.; Tortora, V.; Marin, M.; Souza, J. M.; Castro, L.;
Vila, A. J.; Radi, R. J. Biol. Chem. 2009, 284, 17-26.
Detailed information on our method of electrochemical nitra-
tion of myoglobin and lysozyme and the protocol for reduction/
alkylation of disulfide bonds in lysozyme have been described
previously.38,42-44 Briefly, a water-cooled electrochemical cell with
platinum electrode (lysozyme and cytochrome c) or boron-doped
diamond electrode (lysozyme and myoglobin) was used. After the
reaction was stopped, myoglobin samples were extensively dia-
lyzed.38 We estimate that protein loss during dialysis is <10%. No
further LC separation of nitrated proteins from the unmodified
myoglobin was carried out. Samples were subsequently freeze-
dried and stored at -20 °C for later use. The reaction products
from the electrooxidative nitration of lysozyme were separated
by fast protein liquid chromatography (LC), and reduction/
alkylation of the disulfide bonds was carried out after the LC
separation as described previously.43,44 The electrochemical nitra-
tion of cytochrome c was carried out following the same procedure
as for lysozyme, but the protein solution was ~2.5 less concen-
trated than those of myoglobin and lysozyme. The reaction
products from cytochrome c nitration were LC separated using
the same mobile phases and gradients as in the case of lysozyme,
but the column used was a Hitrap SP sepharose Fast Flow, 5 mL
× 5 mL, from GE Heathcare. After LC separation, the fraction
containing proteins was extensively dialyzed against 10 mM
ammonium acetate (pH ) 5.8) and finally concentrated, as
described previously for lysozyme.43,44
Mass Spectrometry. All tandem mass spectrometry analysis
was performed on a Thermo Finnigan LTQ FT mass spectrom-
eter (Thermo Fisher Scientific, Bremen, Germany). Protein
samples were buffer exchanged in 49.5/49.5% of H2O/CH3OH
and 1% of formic acid using an Amicon centrifugal filter
device, 3 or 10 kDa cutoff (Millipore). The final concentration
was 1-3 μM for myoglobin and lysozyme, for cytochrome c
it was below 1 μM. Samples were directly injected into the
LTQ FT by use of an Advion Biosciences Triversa Nanomate
electrospray source (Advion Biosciences, Ithaca, New York)
at a flow rate of 200 nL min-1. All MS and MS/MS spectra
were acquired in the ICR cell with a resolution of 100 000 at
m/z 400. In total, 150-250 microscans (transients) were
averaged for each fragmentation spectrum. Precursor ions
(single charge state) were isolated in the linear ion trap
(LTQ). The automatic gain control (AGC) target was 2 -10
× 106 ion counts with maximum fill time of2 s. The isolation
width for the ions from the samples of unmodified proteins
and fractions of mono- and bis-nitrated lysozyme was 20-50
Th (single step isolation). Isolation of the ions of nitrated
myoglobin and cytochrome c was achieved in two or three
steps in order to exclude the ions of unmodified protein
present in the sample. The final isolation width was 10-15
Th. Narrowing the isolation width below these values lead
to a serious depletion of the isolated ions and, thus, a much
smaller number of fragments detected. However, we used
narrower five-step isolation with a final width of 5-10 Th
for investigation of neutral losses from nitrated protein ions
upon ECD. CID was carried out in the LTQ with a normal-
ized collision energy of 9-20%, duration of 30 ms, and Q )
0.25. Electrons for ECD were produced by an indirectly
heated barium-tungsten cylindrical dispenser cathode (5.1
mm diameter, 154 mm from the cell, 1 mm off axis) (Heat-
Analytical Chemistry, Vol. 82, No. 17, September 1, 2010 7285