Biology Reference
In-Depth Information
and guidelines are considered for obtaining
sensitive and reliable analysis: the signature
peptide must be unique in the genome and
should contain 7 to 30 amino acids; it should
not be susceptible to post-translational modi
that have isotopic labels but the same sequences
as those for the peptide targets. Two emerging
core strategies for targeted biomarker quantita-
tion are (1) LC-SID-MRM-MS and (2) the combi-
nation of the so-called SISCAPA technology and
quantitative MS, which often is LC-SID-MRM-
MS.
164,165
From a technology viewpoint, these
technologies (and others) really differ by the
methods used for
ca-
tions and missed cleavage sites during enzymatic
digestion. Quantitation of a signature peptide
target in a complex digest preferably requires 2
to 3 transitions,
150,151
although the use of a spiked
reference peptide with stale isotope labels can
relax this requirement.
152,153
Quantitation of the
precursor protein further requires comparable
results from 1 or 2 additional signature
peptides.
152,153
Cautions also need to be paid to
the signature peptide degeneracy of proteins in
complex human proteome samples.
154
Selection of precursor-to-fragment transitions
has been facilitated by
in silico
methods based on
algorithms that use theoretical and empirical
data generated from proteomic analysis world-
wide. Accessible repositories of MS/MS spectral
libraries of peptides, as well as the wide array of
computational tools such as Skyline,
155
AuDIT,
156
ATAQS,
157
mProphet,
158
UIS,
159
AIMS
160
and TIQAM,
161
enable the rapid and
effective prediction, selection, and validation of
peptide MRM/SRM transitions. Commercial
software packages from mass spectrometer
suppliers have also been improved signi
simplifying the
sample
complexity.
MRM MS can quantitatively analyze a large
number of peptide targets in a single experiment.
When some of the peptides are spiked with
isotopic reference peptides, the corresponding
endogenous peptides with the same sequences
can be accurately quanti
ed. Furthermore,
when the absolute amounts of the reference
peptides are known, absolute amounts of the
endogenous peptides can then be calculated.
This capability of MRM MS for absolute quanti-
tation of peptides (e.g., signature peptides for
protein biomarkers) is profound, which allows
for comparable measurements from different
laboratories and on different platforms of tar-
geted proteomic quantitation.
127,166
Another
advantage for using isotopic reference peptides
is the increased con
dence in the MRM MS
signal for counterpart endogenous peptides,
relaxing the requirement for the signature
peptide number for a given protein target. The
isotopic reference can be designed to have very
close elution time to or the same elution time
as that of counterpart peptides.
The protein absolute quantitation (AQUA)
strategy
165
opens the door to global application
of isotopic references for targeted proteomic
quantitation and sets the foundation for the
LC-SID-MRM-MS measurement of signature
peptides of protein biomarkers.
141,167,168
This
technology uses AQUA peptides as quantitation
references. The reference peptides have the same
amino acid sequences as their native counterparts
but with stable isotope labels to introduce
signi
cantly
in recent years. They include MRMPilot
,
Pinpoint and SRM Work
ow, QuantOptimise
and Verify, and MassHunter Optimiser. Online
transition databases such as TraML, Peptide-
Atlas, SRMAtlas, MRMaid, MRMaid-DB,
GPMDB, PASSEL, and QuAD can also facili-
tate high-throughput development of MRM
methods.
153,162,163
Quantitation of Signature Peptides
Although MRM experiments can be used for
direct quantitation of peptide targets, the fast-
growing utilization of these experiments for
quantifying protein biomarkers bene
ts greatly
from the use of internal quantitation standards.
These standards are typically synthetic peptides
cant mass shifts for selective MRM
measurements. They are typically introduced to
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