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concomitant species (e.g., PTMs or allele ratios at
heterozygous loci) are analyzed by top-down
proteomics, expression ratios can be calculated
using spectral intensity ratios.
3,86,87,121
e
123
For
proteins exhibiting complex PTM patterns with
overlapping isobaric species, the ratios of each
unique species may be determined from MS/MS
fragment ion ratios.
122
e
124
Differential alkylation
of amino acid functional groups (e.g., thiols,
amines, and acids) has also been shown for
top-down.
125,126
A number of groups have used
stable isotopes in the cell culture media for protein
quantitation.
85,127,128
For example, recent reports
used
14
N/
15
N metabolic labels on histidine,
leucine, and tryptophan to determine protein
expression ratios in
S. cerevisiae
40
and progressive
methylation of histone H4K20 during the cell
cycle.
129
Collier et al. also applied metabolic
labeling for quantitative comparison of intact
protein expression in
A.
for bottom-up proteomics, as concurrent PTMs
often are not observed on a single tryptic peptide,
requiring analysis of either the undigested protein
or large peptides (
Figure 2
).
26
Site characterization
of combinations of methylations, phosphoryla-
tions, acetylations, and catabolism events has
been performed with top-down in diverse protein
systems.
2,15,17,53
Numerous other PTM classes
have also been characterized. For example, Lour-
ette et al. used high-resolution mass spectrometry
to characterize approximately 250 nitration and
oxidation events on intact calmodulin.
136
Also,
Dorrestein et al. used top-down to unravel steps
in natural product formation by the characteriza-
tion of diverse acyl and peptidyl intermediates
on the carrier domains of nonribosomal peptide
synthesis (NRPS) and polyketide synthases.
137
Extension of top-down to the more complex
patterns of glycosylation has also been demon-
strated, having potentially broad application
for the characterization of protein thera-
peutics.
55,96,133,138
e
142
Analysis of deamidation
events, in which hydrolysis of amino acid side
chain amides results in a 1 Da mass shift, has
also been demonstrated. For example Zabrous-
kov et al. used high-resolution FTMS to study the
rate of stepwise deamidation of
avus
and human em-
bryonic stem cells.
127,130
A variety of label-free
approaches have also been developed. For
example, Roth et al. showed that protein quantita-
tion over a broad linear concentration range with
subfemtomole detection limits from simple
mixtures is possible directly from MS spectra
intensities.
41
For complex mixtures, Julka et al.
combined conventional HPLC with ultraviolet
detection with online MS analysis to quantify
speci
five asparagine
and glutamine sites in ribonuclease A
143
and Li
et al. showed that sequential fragmentation events
(i.e., MS
3
) could differentiate isomeric aspartic
acid and isoaspartic acid products of asparagine
deamidation in aged beta2-microglobulin.
144
c proteins in the complex media demon-
strating good linearity, precision, and accuracy.
131
Differential mass spectrometry (dMS) may be
useful for de
ning expression patterns of proteins
from complex mixtures.
132
For example, dMS
was recently used to characterize changes in apo-
lipoproteins in human high-density lipoprotein
(HDL).
133
Membrane Proteins
Two recent reviews discussed intact mass
pro
ling with MS/MS analysis on diverse
membrane proteins.
145,146
Membrane proteins
are thought to constitute approximately a third
of the proteome and are often associated with
cellular signaling and therapeutic drug interac-
tions.
147
Analysis of membrane proteins by
top-down may improve understanding of
ligand
e
receptor interactions, probe interac-
tions between proteins and lipids, and help
PTMs
Highly modi
ed proteins are commonly asso-
ciated with biological processes such as micro-
tubule formation,
134
gene regulation,
89
neuronal
myelination,
135
anddisease.
134
Obtaining concom-
itant PTM information is typically challenging
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