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characterize closely related proteins or large
polypeptides derived from limited proteolysis
(middle-down MS
15
). This chapter describes the
key concepts and the maturation of technologies,
sample preparation approaches, and informatics
tools that are important for streamlined MS
and MS/MS analysis of proteins using high-
resolution
Indeed,
the most recent reports have shown
potential
cation of thousands of
proteins from human cells.
22
Although top-down
and bottom-up have been presented as competi-
tive strategies, comparative studies have shown
that the methods provide complementary infor-
mation with small and larger proteins often
over-represented with top-down and bottom-up,
respectively.
23
e
25
However, top-down may best
complement peptide-based analyses, which typi-
cally don
for
identi
and high-mass
accuracy mass
spectrometers.
5,6,15
e
18
t provide 100% amino acid sequence
coverage without redundant processing with
different enzymatic and chemical cleavage strate-
gies (
Figure 2
).
26
Poor sequence coverage can limit
the ability of bottom-up to differentiate expres-
sion patterns of gene family members
27
or hetero-
geneous modi
'
Top-Down and Bottom-Up Proteomics
Intact mass analysis of proteins, often followed
by MS/MS, protein identi
cation from genome
predicted databases, and localization of PTMs,
provides the basis for most high-resolution top-
down MS work
ows (
Figure 2
).
5,19
From the
context of comprehensive analysis of proteins
across an entire proteome (i.e., proteomics),
top-down is often compared with bottom-up
methods that use proteases (e.g., trypsin) to
digest proteins into manageable peptides
(1
e
3 kDa) prior to MS and MS/MS.
20
In
bottom-up, MS and MS/MS data are used to
deduce peptide sequence information for the
identi
cation states,
28
making studies of
splicing events and simultaneous PTMs extremely
challenging in large-scale bottom-up proteomics
projects.
26
MASS SPECTROMETRY
H
ARDWARE FOR TOP-DOW
N
Ionization
Critical to analysis by mass spectrometry is
the presence of a charged analyte. Traditional
methods for producing charged molecules (ion-
ization) require analyte present in the gas phase
prior to analysis. Given the inherent low volatility
of polypeptides, the routine analysis of intact
proteins with MS was limited until the advent
of electrospray ionization (ESI)
29
and MALDI
(
Figure 3
).
30,31
These soft ionization techniques
introduce analyte into the gas phase while ioniz-
ing analyte through proton exchange under condi-
tions that do not break labile amino acid bonds.
32
Both ESI and MALDI are suitable for achieving
identi
cation of the parent protein from a gene
database. Development of bottom-up methods
for high-throughput protein identi
cation, quan-
titation, and PTM analysis has outpaced top-
down methods over the last decade because of
the relative ease with which small peptides can
be analyzed with MS.
21
Compared to bottom-up, top-down has pri-
marily been applied for targeted, hypothesis-
driven experiments on relatively simple protein
systems. Global proteomics projects have been
limited due to technical challenges related to
sample preparation, chromatography, ionization
of proteins with diverse physiochemical pro-
perties over broad concentration ranges, and
automation of instrument hardware and
data interpretation. However, the promise of
abird
cation and structural determination of
peptides, proteins, oligonucleotides, lipids, and
synthetic polymers over a range from hundreds
to several hundred thousandDa.
32
Further discus-
sion in this chapter predominantly focuses on ESI
because of its role in high-resolution MS analysis
s-eye view for an entire proteome has
spurred signi
'
cant improvements in the scale
and throughput of modern top-down work
ows.
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