Biology Reference
In-Depth Information
INTRODUCTION
peak capacity available from the combined
separations and MS technologies severely limits
the ef
Advancement in Mass Spectrometry
Continuous progress in mass spectrometry
and polypeptide separations has made proteo-
mics analysis more sensitive and comprehen-
sive. Hundreds
ciency and overall sensitivity achieved.
Additionally, each separation or enrichment
step introduces multiple fractions, each of
which must be analyzed, and this step also
introduces variability that renders quantitative
proteomics more dif
and even thousands
of
cult and challenging.
5
Another major problem limiting shotgun
proteomics is the data-dependent acquisition
(DDA) mode generally used by mass spectrom-
eters for automated ion selection. With the
DDA method to ion selection, the most abun-
dant ionized species from each MS survey
scan is selected for subsequent isolation, activa-
tion, and tandem mass spectral analysis.
1
Orig-
inally, this process greatly facilitated proteome
coverage and extended dynamic range of detec-
tion for shotgun proteomics. A companion
method referred to as
proteins are routinely identi
ed
from complex mixtures such as tissue or
cellular extracts, organelles, bacteria, and body
ed and quanti
fluids.1
1
In spite of this progress, complete char-
acterization of a proteome remains a challenge
today due to factors such as the large dynamic
range of protein expression, post-translational
modi
cations (PTMs), the complexity of rela-
tive stoichiometries between proteins, as well
as lack of methods to amplify proteins as poly-
merase chain reaction (PCR) does for DNA. It is
generally accepted that the dynamic range
problem may be solved by sample fractionation
and/or enrichment. Thus, most laboratories
engage in numerous strategies to separate
proteins and/or peptides prior to mass spectro-
metric (MS) analysis using methods such as
anion or cation exchange, one- or two-
dimensional polyacrylamide gel electropho-
resis, isoelectric focusing, and af
”
that attempts to prevent reselection of the
same ion over and over again added further
bene
“
dynamic exclusion
ts to coverage and dynamic range.
6
However, this addition to the automated ion
selection process also fails to provide full
coverage of peptides in a complex mixture as
judged by the numerous studies showing the
nonreproducible nature of peptides detected
in replicate analyses of the same sample.
7
One
MS method that provides some additional
peak capacity is gas-phase fractionation (GPF).
With GPF, the sample is repetitively injected
into the mass spectrometer and a unique m/z
range is used for data-dependent precursor
ion selection during each replicate liquid
chromatography
e
mass spectrometry (LC-MS/
MS) analysis of a given sample.
8,9
However,
as we have recently shown, although optimum
m/z ranges for GPF may be predicted from the
genome of the considered species to maximize
proteome coverage (i.e., genome-based GPF),
the number of new protein identi
nity capture
or depletion.
2
e
4
Ideally, these methods should
be orthogonal to each other to provide the
best peak capacity and ensure that each fraction
is as unique as possible. In a perfect case, each
fraction would then be thoroughly character-
ized by MS analysis such that all components
present could be detected. However, in practice
none of the available pre-MS separation tech-
niques provide suf
cient resolution to simplify
the mixture prior to MS analysis for complete
characterization. Thus, the continuing problem
for shotgun proteomics is that often the same
peptide is found in multiple fractions and
analyzed by MS multiple times, which is
inef
cations still
reaches a plateau using all three of these MS
methods combined.
10,11
cient, and too many unique peptides co-
elute for the MS to acquire tandem mass spec-
tral data on all of them. This lack of suf
cient
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