Biology Reference
In-Depth Information
characterize novel biomarkers of disease.
146
Membrane proteomics has been challenging due
to strong hydrophobicity and insolubility in
RPLC-compatible buffers; however, investi-
gations over the last decade have helped de
of closely related protein isoforms and PTMs in
disease pathobiology and highlight the poten-
tial of high-resolution mass spectrometry in
clinical diagnostics.
ne
methods
cation
of integral membrane proteins intended for top-
down analysis.
81,148
In these studies, top-down
has facilitated the characterization of numerous
PTM classes,
for
solubilization and puri
CONCLUSION
During the systems biology era, great
emphasis has been placed upon large-scale
processing of full proteomes to facilitate charac-
terization of vital parts of living systems
including their response to disease. Over the last
decade, top-down MS has matured at a slower
pace compared to peptide-based work
including proteolytic processing,
disul
de bonds, cysteinylation, and covalent
heme modi
cation.
145
Tissues and Bio
uids
Analysis of tissues and humoral
ows.
However, continued technology advancements
that improve speed, sensitivity, resolution, and
dynamic range result in increased understanding
of how to analyze proteins that are present in
heterogeneous states with diverse physiochemi-
cal properties. The top-down approach provides
peak capacities that allow characterization of
thousands of proteins in reasonable time frames.
Top-down is also a valuable tool for routine
protein molecular diagnostic applications in
which protein heterogeneity is categorized with
speci
fluids (e.g.,
plasma, blood, and cerebrospinal
fluid) presents
unique challenges for proteomics investigations
due to the presence of highly abundant proteins
and the large dynamic range of protein concen-
trations.
149
Despite these challenges, investiga-
tions have begun to highlight the potential of
top-down MS in analyzing proteins derived
from tissues and bio
uids. For example, Roth
et al. used top-down proteomic analysis to char-
acterize diverse PTMs and SNPs on proteins
from leukocytes isolated from human blood.
3
Work has shown that cTnI phosphorylation
may serve as a biomarker of disease progres-
sion leading to chronic heart failure.
4,150,151
Mazur et al. characterized differential glycosyl-
ation of apolipoporotein C-III from plasma
HDL for healthy and cardiovascular disease
patients.
133
Also, Nepomuceno et al. reported
that high-resolution accurate mass pro
city not attainable with conventional
molecular biology techniques or bottom-up MS.
Perhaps most important is that the proteomics
field is entering a phase in which top-down and
bottom-up experiments that at one time were
possible only on a single type of instrument are
now possible on a variety of systems, suggesting
that the scienti
c community will be able to better
integrate top-down MS into their protein charac-
terization toolbox.
ling
with FT-ICR MS could characterize most of
the over 100 known protein mutations observed
on intact transthyretin isolated from blood.
152
Subsequent work by Theberge et al. has gener-
ated transthyretin amyloidosis and hemoglo-
binopathy clinical assays on an Orbitrap mass
spectrometer with automated sample introduc-
tion, MS/MS events, and data analysis proto-
cols.
153
These cumulative efforts highlight the
strength of top-down MS for quantifying ratios
References
1.
Jungblut PR, Holzhutter HG, Apweiler R, Schluter H.
The speciation of the proteome.
Chem Cent J
2008;
:16.
2. Kellie JF, Tran JC, Lee JE, et al. The emerging process
of top down mass spectrometry for protein analysis:
biomarkers, protein-therapeutics, and achieving high
throughput.
Mol Biosyst
2010;
2
6
:1532
e
9.
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