Biology Reference
In-Depth Information
(A)
(B)
300
120
PSAQ standard
y
=
1.08x
R
2
=
0.9900
PSAQ standard
y
=
L05x
R
2
=
0.9924
100
250
80
200
QconCAT standard
y
=
0.58x
R
2
=
0.9952
150
60
QconCAT standard
y
=
0.19x
R
2
=
0.9826
100
40
AQUA standard
y
=
0.21x
R
2
=
0.9805
50
20
AQUA standard
y
=
0.04x
R
2
=
0.7580
0
0
0
20
40
60
80
100
0
50
100
150
200
250
Added SEA (ng/ml urine)
Added TSST-1 (ng/ml urine)
FIGURE 5
The comparison of concentration curves of peptides NVTVQELDLQAR (SEA) and LPTPIELPLK (TSST-1) in
SEA (A) and TSST-1 (B) spiked urine samples obtained using different isotope-labeled internal standards: synthetic peptides
(AQUA) or generated from QconCAT or PSAQ. Each data point is the mean value
S.E. of three analytical replicates.
(Reproduced with permission from reference #31).
isotope-labeled recombinant protein used in clin-
ical sample settings, quanti
at different sites could cause signi
cant data
cation method of uri-
nary albumin with a clinically relevant dynamic
range of approximately 3 mg/L to 300 mg/L
using 15
N
-labeled recombinant human serum
albumin (HSA) internal standard was developed
and its clinical performance for 138 patent
samples comparable to a commercially available
immunotubidometric assay was demon-
strated.
49
It has also been demonstrated recently
in an inter laboratory SISCAPA MRM-MS
study that stable isotope-labeled internal
protein standard improved precision by 5%
while doubling the assay accuracy.
34
However,
isotope-labeled recombinant proteins have not
yet been applied in any large-scale biomarker
veri
variation.
BIOINFORMATICS SOFTWARE
FOR MRM-MS ASSAYS AND
B
IOMARKER VERIFICATIO
N
With the large amount of potential
biomarker candidates to be veri
ed, setting
up an MRM-MS assay for each peptide with
multiple transitions manually could become
time consuming (and confusing), especially
when time-scheduled transitions also need to
be set up to increase both throughput and
sensitivity. In addition, once high-throughput
multiplexed MRM-MS assays
cation studies, probably due to the time
and expense required for
for parallel
their generation.
biomarkers
cation are optimized, data
processing must be automated as well to
improve throughput and minimize error.
Bioinformatics tools to facilitate both the
earlier stage surrogate peptides determination
and the later stage data processing require
continued development. Both commercial and
open source software have been developed in
'
veri
As the technology for
recombinant protein
expression
cation continues to
develop, isotope-labeled recombinant proteins
could become a very useful tool for later targeted
biomarker veri
and
puri
cation efforts, especially when
the efforts are performed across multiple
sites where
sample preparation procedure
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