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is a 148 kDamonoclonal antibodyon a 9.4TFT-ICR
hybrid mass spectrometer.
47
Despite the importance of FTMS instruments in
the development of early top-down platforms,
their high cost and slow acquisition rates (
reduction approach on a linear QIT for the top-
down characterization of
E. coli
70S ribosomal
proteins.
51
Similarly, Liu et al. used ion/ion reac-
tions in a Q-TOF to characterize unknown
proteins with novel PTMs from
E. coli
.
58
1
sec) limit MS/MS spectral quality and duty
cycle during online applications, although new
high-
>
Tandem Mass Spectrometry
Mass spectrometers support a variety of
MS/MS approaches that can be categorized as
either threshold or nonergodic dissociation
techniques.
19,59
Threshold dissociation methods
impart energy through collisions
60,61
and
photons,
62
resulting in vibrational heating and
fragmentation at the weakest bonds. Nonergodic
techniques impart energy through the capture of
a low-energy electron, resulting in fragmentation
directly at the site of electron capture.
63
Threshold approaches such as collisionally acti-
vated dissociation (CAD)
60
or collision-induced
dissociation (CID),
61
nozzle/skimmer dissocia-
tion (NSD),
64
and infrared multiphoton dissocia-
tion (IRMPD)
62
are the most widely exploited
for top-down work
eld instruments and enhanced FT-mode
processing platforms demonstrate improved
sampling rates, resolution, mass accuracy, and
mass range for proteomics work
ows.
44,48
e
50
Also, new commercial MALDI-TOF, ESI-TOF,
and ESI-QIT mass spectrometers have substan-
tially improved resolving powers (
50,000) which
promise to expand their utility for high-resolution
analysis of proteins approaching 30 kDa.
44
Simi-
larly, investigators have sought to use the speed
and sensitivity of Q, QIT, and TOFmass analyzers
to facilitate high-throughput MS/MS experiments
on intact proteins to permit protein identi
cation
without accuratemass determination on the intact
molecules.
23,24,51,52
For example, Madsen et al.
have developed novel MS/MS techniques on
QIT instruments that permit the detection of
a large number of fragment ions for protein
identi
ows. Fragmentation with
threshold approaches predominately occurs at
protein amide bonds, forming N- and C-terminal
fragment ions (denoted as b and y ions, respec-
tively; see
Figure 4
A).
65
However, activation of
all the molecular vibrational/electronic degrees
of freedom can lead to preferential fragmentation
pathways and deleterious fragmentation events
such as loss of water or secondary fragmentation
(i.e., fragmentation of fragment ions) that result
in fragment ions that do not contain either the
N- or C-terminus of the protein.
66
In threshold
approaches, the composition and extent of frag-
mentation is known to depend on the precursor
mass and charge, which allow tuning of frag-
mentation parameters, including precursor ion
internal energy, activation time, and activation
energy. Automated decision-tree methods have
been designed to optimize fragmentation
types and conditions during online applica-
tions.
39,67
For example, Patrie et al. showed that
twofold increase in fragment ion matches could
cation.
53
Additionally, Ginter et al. demon-
strated that MS
3
events enable
de novo
sequenc-
ing of portions of protein sequences, with the
resulting amino acid sequence-tag used to identify
the protein observed.
54
One notable distinction between MS/MS on
proteins versus peptides is that a protein
MS/MS spectrum is typically populated by
highly-charged fragment ions with molecular
weights of greater than 5 kDa. Misassignment
of fragment ion charge on low-resolution instru-
ments can result in improper fragment ion mass
assignment, reducing con
dence in downstream
protein assignments, as discussed later in this
chapter.
55
To overcome this challenge, groups
have developed gas-phase ion/ion reaction
schemes that convert multiply charged MS/MS
product ions into singly charged species, facili-
tating correct mass assignment.
56,57
For example,
Chi et al. demonstrated the utility of the charge
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