by fragmentation MS/MS methods, such as collision induced
dissociation (CID).11,12 However, MS/MS analysis of protein
digests (the “bottom-up” approach) allows the possibility that
peptides with PTMs may be missed due to poor or insufficient
separation on the HPLC column or poor ionization efficiency. The
alternative approach is “top-down” protein characterization, in
which intact proteins are ionized and subsequently analyzed in a
mass spectrometer.13-17 The complexity of the resulting mass
spectra requires a high-resolution technique, such as Fourier
transform ion cyclotron resonance (FT-ICR) mass spectrometry.
The key advantage of the top-down approach is that the measured
mass of an intact protein may be compared with the mass
calculated from the known protein sequence, thus providing
information about all the modifications present in the protein and
retaining connectivity between the modifications. In the case of
multiple modifications, several mass peaks related to protein
isoforms may be present in the mass spectra, and their relative
intensities provide information on the extent of each of the
modifications. MS/MS fragments of intact proteins can be
analyzed to localize the sites of modifications in a similar way to
that for peptides from protein digests. The top-down method has
been demonstrated on proteins up to ~200 kDa.17 The downside
of this method is that the time of MS/MS analysis is longer than
in the bottom-up approach, due to a much larger number of
fragmentation channels.
Several MS/MS fragmentation techniques are available for both
bottom-up and top-down approaches. “Slow-heating” methods,18 such
as CID or infrared multiphoton dissociation (IRMPD),19,20 utilize
different techniques to excite and dissociate molecular ions
thermally. CID and IRMPD produce heterolytic cleavages of the
amide bonds in the polypeptide chain giving rise to b and y
fragment ions that contain the N- or C-terminus, respectively.21
As thermal methods, they cleave the weakest bonds first. Since
its introduction in 1998, electron capture dissociation22 (ECD) and
its sister method, electron transfer dissociation (ETD),23 have also
become popular in MS/MS studies. ECD is a fast fragmentation
technique whereby cleavage of the peptide backbone occurs
following low-energy (<0.2 eV) electron capture and the subse-
quent cascade of intramolecular radical-driven reactions, the
precise pathways of which are still of some discussion.24-27 ECD
cleavage of the N-CR bonds occurs producing mainly N-
terminus c' and C-terminus z• (or c• and z') fragment ions.
One of the advantages of ECD over the thermal methods is
that it provides a more uniform pattern of cleavages along the
(13) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson,
E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812.
(14) Kelleher, N. L. Anal. Chem. 2004, 76, 196A-203A.
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backbone, with the only exception being cleavage of the
N-terminal to proline28 and thus leads to greater peptide
sequence coverage.29,30 Unlike the thermal methods, disulfide
bridges are efficiently cleaved by ECD of peptides,24 and following
capture of a second electron, fragments from the peptide segment
inside the “disulfide loop” can be produced. Furthermore, ECD
fragments retain labile post-translational modifications,31 while
CID and IRMPD tend to cleave them. Examples of the efficient
use of ECD for localizing PTM sites include phosphorylation,32,33
N- and O-glycosylation,34,35 ubiquitination,36 sumoylation,37 and
others. Nevertheless, there have been observations that ECD is
not universally efficient for all possible peptide modifications. We
have recently demonstrated that addition of nitration to tyrosine
severely inhibits the production of ECD sequence fragments in
peptides.38 A similar effect was reported by the Beauchamp group
for benzyl modifications of cysteine which have an electron affinity
(EA) of g1.00 eV.39 Specifically, 3-nitrobenzylcysteine (EA ) 1.00
eV) and 3,5-dinitrobenzylcysteine (EA ) 1.65 eV), termed “elec-
tron predators”, inhibit peptide backbone cleavage by ECD and
the related electron transfer dissociation (ETD) completely.39
Apparently 3-nitrotyrosine, structurally similar to nitrobenzylcys-
teine, was also acting as an “electron predator” in our ECD
experiments. However, we demonstrated that ECD of the triply
charged nitrated peptides resulted in some singly charged
sequence fragments, which may be the products of secondary
electron capture.38 That result indicated that top-down ECD of
intact nitrated proteins may be efficient, as multiple electron
capture by multiply charged protein ions usually occurs,24,30 the
hypothesis which we put to test in this work.
In this study we optimize and compare top-down ECD, CID,
and IRMPD of nitrated proteins: myoglobin, cytochrome c, and
hen egg white lysozyme (HEWL). Our choice of proteins was due
to the different behaviors of their un-nitrated forms under ECD.
Previously, we have shown that z > 14+ cations of unmodified
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(33) Stensballe, A.; Jensen, O. N.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A.
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7284 Analytical Chemistry, Vol. 82, No. 17, September 1, 2010