Biology Reference
In-Depth Information
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O
18
O,
labeling, which results in mixed labels
attractive technique for biomarker veri
cation
thereby affecting O
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/O
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ratios.
and possibly even validation.
In general, SRM assay includes the following
steps: digestion of proteins, LC separation of
peptides,
Chemical Labeling
Approaches to chemical labeling of proteomic
samples use heavy or light isotope-labeled and
chemically reactive tags. For instance, isotope-
coded af
ionization of peptides with ESI,
first quadrupole, frag-
mentation of peptides in the second quadrupole,
filtering of peptides in the
filtering of peptide fragments in the third quad-
rupole, and measurement of intensities of three
selected fragment ions.
96,97
A known amount
of a heavy-isotope labeled peptide is often
spiked into the digest and used to calculate the
absolute amount of
nity tags (ICAT) allow for labeling of
cysteine residues in proteins.
93
Once labeled,
proteins
from both groups are combined,
af
ed through biotin tags, and
peptides with heavy and light labels are quanti-
nity-puri
the endogenous
light
peptide. Addition of
stable-isotope labeled
fied based on their differential MS1 signals.
Exclusive labeling of cysteines is the main limita-
tion of this approach, as it reduces protein
sequence coverage. On the other hand, due to
the af
peptide standards
increases
speci
city and
reproducibility of quanti
cation due to the
correct identi
cation of analyte peak in the pres-
ence of multiple contaminant peptides and
accurate relative quanti
nity capture of these peptides, sample
complexity is signi
cation. It is sometimes
accepted that trypsin digestion proceeds to full
conversion and that the amount of proteotypic
peptide re
cantly simpli
ed, which
facilitates
quanti
cation
of
low-abundance
proteins.
Isobaric tags for relative and absolute quanti-
ects the absolute amount of the cor-
responding protein. Such an assumption is not
always correct but is acceptable when the rela-
tive abundance of proteins is measured. More
accurate measurement of absolute protein
amounts is achieved with heavy isotope-
labeled proteins
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or concatenated peptide stan-
dards,
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which account for variation of protein
digestion.
With state-of-the-art SRM assay, up to 100
peptides representing 100 medium-to-high-
abundance proteins in the range 0.1
fication (iTRAQ)
94
or tandem mass tags (TMT)
95
are amine-reactive tags that produce reporter
ions upon MS/MS peptide fragmentation.
Following protein digestion, iTRAQ allows for
peptide labeling in up to eight different biolog-
ical conditions. Following labeling, peptides
from all conditions are pooled together and
analyzed by LC-MS/MS (
Figure 3
). Unlike other
labeling approaches, iTRAQ utilizes MS/MS
spectra for relative quanti
cation.
75
g/mL to
1 mg/mL can be measured simultaneously in
the unfractionated digest of biological
m
uid
Selected Reaction Monitoring Assays
Selected reaction monitoring (SRM) is
a quantitative analytical assay performed on
a triple-quadrupole, quadrupole-iontrap, or
quadrupole-TOF mass spectrometer. Although
protein identi
while achieving coef
cients of variation under
20%.
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There are several concerns with
SRM-based assays, and these mostly stem from
sample complexity and limitations in instrument
sensitivity and selectivity. Ideally, the number of
sample preparation steps prior to LC-SRM
measurement should be minimal in order to
allow for high-throughput analysis and mini-
mize variability, although this bene
cation approaches are tuned to
identify thousands of proteins in a limited
number of samples, SRM assays are intended
to measure a very limited number of proteins
in a large set of samples. This makes SRM an
t comes at
the cost of decreased assay sensitivity.
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