Biology Reference
In-Depth Information
signi
cant work is still required to bring these
methods to general acceptance.
proteins from small amounts of material. For
example, Roth et al. recently showed that 343
protein species could be reproducibly character-
ized from 5
10
4
HeLa cells with less than 30
min analysis time.
41
This study also showed that
detection of common modi
CURRENT STATUS
cation states and
Top-Down Work
ows
Efforts to streamline top-down acquisition for
members from all
five of the histone families
may be routinely possible from a few thousand
cells with 5 min elution gradients.
41
“
”
applications have occurred within the
last decade with the development of algo-
rithms for spectra deconvolution,
105,106
infor-
matics tools for protein database retrieval,
100
and automation of mass spectrometers.
107
Initial
top-down proteomics
omics
Quantitation
Monitoring protein abundance changes or
occurrence of PTMs is necessary for classifying
developmental stages or disease courses. Many
MS methods have been developed for bottom-up
quantitation. For relative quantitation, metabolic
labeling such as stable isotope labelingwith amino
acids in cell culture (SILAC) has widespread
application when proteins are derived from
cell cultures or animal models.
112,113
investigations
largely
exploited of
ine multidimensional separations
for the characterization of microorganisms
and human cells.
3,39,100
However, the transition
to online LC/MS has dramatically increased
throughput,
40
with recent examples highlighting
the feasibility of high-resolution top-down MS
for large-scale and sensitive proteomics applica-
tions. For example, Tran et al. recently used IEF,
GE, nano-RPLC, and FTMS to identify 1,043
gene products (
Proteins
from noncultured samples (e.g., bio
uids or
tissues) can be labeled enzymatically by using
18
O-labeled water during proteolytic digestion,
114
or by the addition of chemical tags at thiols or
amines
105 kDa) from human HeLa S3
cells that were dispersed into more than 3,000
protein species.
22
Similar studies on H1299
human lung cancer proteins identi
such as
isotope-coded af
nity tags
(ICAT),
115
ed approxi-
mately 690 gene products with about three times
as many protein species identi
isobaric tags for relative and absolute
cation (iTRAQ),
116
or tandem mass tags
(TMT).
117
For absolute quantitation (AQUA),
stable isotope labeled synthetic peptides are used
as internal standards.
118
Alternative
quanti
ed for each gene
product.
108
These results help highlight
the
degree to which human genes are diversi
ed by
PTMs, RNA splicing, and catabolism events,
and the role that top-down may play in rational-
izing these events via proteome-wide initiatives.
Next-generation tools may continue to provide
improved dynamic range, mass range, and
throughput for top-down data processing plat-
forms (e.g., larger superconducting magnets
with FT-ICR, high-
”
approaches include comparisonof relative intensi-
ties/peak areas for precursor ions or their frag-
ment ions such as selected reaction monitoring
(SRM) as well as numbers of MS/MS spectra
(spectral counting).
119
Similar quantitation strategies for intact
proteins have recently been reviewed.
120
Single-
point mutations and PTMs have been shown to
have a minimal effect on LC elution time, MS
response, and fragmentation patterns at the intact
protein level, making direct comparison of expres-
sion ratios fromMS intensities feasible for related
species present concurrently in a MS spectrum.
For example, reports have shown that when
“
label-free
eldOrbitrap instruments, and
enhanced FT data-processing modes).
48
e
50,108
e
111
Additionally, the improved peak capacity
observed with monolithic and SP separations are
also poised to streamline top-down platforms by
minimizing LC/MS gradient durations and
improving detection limits for 1D processing of
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