Biology Reference
In-Depth Information
proteomics.
98
For top-down, the web-based
ProSightPTM and its commercial analogue,
ProSightPC
quantities of proteins (0.1
e
5mg)priortosubse-
quent LC/MS/MS. Online electrophoretic
separations such as capillary isoelectric fo-
cusing (CIEF), capillary zone electrophoresis
(CZE), and capillary electrochromatography
(CEC) have been used for top-down MS to
characterize diverse protein systems includ-
ing microorganisms, bio
, are widely available protein
search engines.
99
ProSightPTM uses a Poisson
model to assess the statistical signi
cance of
observedMS/MS fragment ion matches to theo-
retical fragment ions and computes a measure
of con
uids, protein
e
ligand
interactions, biopharmaceuticals, and dietary
proteins.
90
e
92
In-solution size-based separations using size
exclusion chromatography (SEC) and gel-
electrophoresis (GE) are also used for top-
down. In SEC, proteins migrate through a porous
polymeric column being separated by their
hydrodynamic volume, with larger proteins
eluting more rapidly than smaller proteins.
SEC has been used to characterize PTMs and
mispredicted signal peptides on thylakoid
lumen proteins from
A. thaliana
95
and to identify
membrane protein complexes in
Synechocystis.
80
Also, Mazur et al. used SEC with LC/MS/MS
to characterize therapeutic monoclonal anti-
bodies and their breakdown products.
96
Solution-based GE devices
97
exploit continuous
dence in protein assignment. The proba-
bility of a spurious fragment
ion match is
largely de
ned by fragment ion mass accuracy
and the number of experimental fragment ions
matched to a protein in a database relative to
the total number of fragment ions observed.
100
The inclusion of protein molecular mass into
the search improves search times by limiting
the search space to a window about the
observed molecular weight. To account for
PTMs, ProSightPTM uses one of two strategies:
(1)
-annotated databases that include
all known or predicted PTMs
101
“
shotgun
”
and (2) an
error-tolerant
m-mode
that
assigns fragment ions that contain a PTM to
a theoretical protein if the mass defect associ-
ated with the PTM on the fragment ion is also
observed at
search called
D
the intact protein level.
99
For
flow gel elution to separate proteins on a poly-
acrylamide column and can separate milligram
quantities of proteins across broad mass ranges
(5
e
200 kDa) with high chromatographic resolu-
tion.
75
Recent investigations have combined IEF,
GE, and LC/MS into a three-dimensional top-
down platform with theoretical peak capacity
of greater than 100,000 and demonstrating the
largest dynamic range for top-down on mamma-
lian cell lysate reported to date.
22,97
unknown modi
cations that cause the mole-
cular weight of the protein to fall outside
a normal search window, the
m-mode
can
be combined with sequential searches across
the database. In the past,
D
this method has
been useful for the identi
cation of proteins
that were subjected to biologically relevant
proteolysis.
39
Alternative informatics approaches have been
proposed for top-down investigations, including
a spectral alignment algorithm that identi
es
protein forms presenting with concomitant
PTMs.
102
BIG-MASCOT extends the working
mass range of MASCOT to handle intact pro-
teins.
103
The precursor ion-independent top-down
algorithm (PIITA) cross-correlates deconvoluted
MS
2
spectra to theoretical MS
2
spectra.
104
These
new search schemes have great potential for
enhanced computational throughput and speci-
INFORMATICS
cation requires informatics
tools that provide statistically signi
Protein identi
cant corre-
lation between MS/MS data sets and predicted
gene products housed in a theoretical database.
Mascot, Sequest, and X!Tandem are commonly
used protein identi
cation tools for bottom-up
ficity
for
top-down
informatics,
although
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