of fragments from the intact nitrated proteins among the three
MS/MS methods tried: ECD, IRMPD, and CID. Although ECD
produced fewer cleavages in the vicinity of the nitration site than
IRMPD, there were sufficient numbers of ECD fragments which
contained the site of nitration. However, if other modifications,
such as multiple disulfide bonds or a heme group, are present,
ECD of the intact protein could be further inhibited. The most
appropriate approach for the top-down MS/MS analysis of intact
nitrated proteins seems to be a combination of different methods
of fragmentation following the reduction and alkylation of any
disulfide bonds present.
ACKNOWLEDGMENT
The Wellcome Trust (Grants 074131 and 080998) (H.J.C. and
V.A.M., respectively), the Ramon y Cajal Programme (Grant
CTQ2006-14959), and the Ministerio de Ciencia y Tecnologia
(Grant CTQ2007-62345), Spain (J.I.), are acknowledged for
funding.
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.
org.
Received for review May 5, 2010. Accepted July 14, 2010.
AC101177R
7292 Analytical Chemistry, Vol. 82, No. 17, September 1, 2010