Biology Reference
In-Depth Information
results obtained using MRM
3
correlated very
well with the ELISA tests. Besides the previ-
ously mentioned strategy, Hossain et al.
38
reported that modi
STABLE ISOTOPE-LABELED
IN
TERNAL STANDARDS US
ED
cation of a triple quadru-
As demonstrated in the literature, stable
isotope dilution is key to reproducibility, accu-
racy, and transferability of MRM-MS-based
assay performance when applied in multisite
studies to establish the applicability of such an
MS-based approach in clinics.
3
e
6
The stable
isotope-labeled internal standards greatly assist
in reducing technical as well as instrumental
variations. There are mainly three types of stable
isotope-labeled internal standards used in
MRM-MS assays to date. A comparison of the
introduction points of these three types of
isotope-labeled internal standards is shown in
Figure 4
.
Stable isotope-labeled peptides of the target
proteins synthesized by chemical methods
(also referred to as AQUA peptides for absolute
quanti
pole mass
sionsou eby
coupling a multicapillary inlet/dual electrody-
namic ion funnel interface improved ion trans-
mission ef
spectrometer
'
ciency that increased MRM peak
intensities from 20- to 150-fold, depending on
the individual peptides. Further testing of the
interface demonstrated a 10-fold improvement
of LOD to the 40 to 80 ng/ml range for the
proteins spiked in nondepleted mouse plasma
with much better reproducibility. Further
advancements of mass spectrometer technology
both from ion transmission ef
ciency and back-
ground noise reduction show great potential to
improve both sensitivity and selectivity of the
MRM-MS-based approach for reliable low-
abundance (ng/ml) biomarker quantitation.
These instrument-based improvements could
be applied to achieve the sensitivities needed
for protein biomarker veri
cation) are the most commonly used
internal standards for MRM-MS assays. It is
also the only applicable internal standard when
absolute quanti
cations either sepa-
rately or in combination with sample enrich-
ment strategies.
ed peptides
are concerned as long as the modi
cation of modi
cation can
FIGURE 4
Typical entry point of
AQUA peptides, QconCAT concate-
mers, and isotope-labeled recombinant
proteins as
Sample
(e.g. plasma, urine)
isotope-labeled recombinant standards
(e.g. PSAQ)
internal
standards
for
quanti
cation of protein biomarkers in
sample processing procedure.
Sample preparation for enrichment or
any other purpose at protein level
QconCAT concatamer
Enzyme digestion
AQUA peptides
Sample enrichment or preparation at peptide
level (SISCAPA, sample cleanup etc.)
MRM-MS analysis
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