Biology Reference
In-Depth Information
peptide libraries
14
of the preliminary peptide
surrogate targets could also be an excellent and
much cheaper alternative for assay development,
especially when many proteins are involved.
Figure 2
shows a general work
such peptides if warranted, what peptide
sequence to use obviously will not be a choice,
although enzyme choice might be another
parameter to consider.
flow to develop
peptide MRM-MS assay for protein quantitation.
Normally, three to
MRM-MS ASSAY PERFORMANCE
CHARACTERISTICS FOR
B
IOMARKER VERIFICATIO
N
five peptides per protein after
method development and optimization are used
to quantify the protein biomarker.
Although the above-mentioned guidelines for
MRM assay development are useful, it is obvious
that in some cases these
ca-
tion, the assay performance of the targeted
MRM-MS assay including sensitivity, speci
To be used for quantitative biomarker veri
“
rules
”
need to be
city,
reproducibility, linear dynamic range, and multi-
plexilbility, as well as potential throughput must
be evaluated and established in a matrix similar
to that being used in the clinical study. The vari-
ance of assay performance at different sites
should also be evaluated if the MRM-MS assay
is to be used in clinical settings. A recent publi-
cation by Addona et al.
3
reported a multisite
(eight sites) systematic assessment of the perfor-
mance of MRM-MS targeted assays applied to
broken to target a speci
c site of interest. Post-
translational modi
cations (PTMs) including
phosphorylation, glycosylation, and acetylation
as well as other modi
cations have been known
to play pivotal roles in cell signaling and recent
evidence also shows that they are functionally
related.
15,16
Therefore, modi
cation quantitation
status at speci
c sites could very well be poten-
tial biomarkers as has been shown in the
scienti
c and medical literature.
17
e
19
When
dealing with MRM-MS assay development for
Potential candidates identified in discovery phase
Potential candidates reported in literature
Potential candidates implicated in pathway analysis
Biomarker protein candidates
to be quantified
Peptides and transitions observed empirically
and/or generated
in silico
Unique to proteome
~8-25 amino acids (mass spec and LC friendly)
Avoid known post translational modification sites
Avoid easily modifiable amino acids
No ragged end
Preliminary peptide surrogates and
transitions lists
Instrument parameters optimization
Peptides with best ionization transitions provide best
intensity and specificity (least interference)
Refined peptides and transitions lists
Peptides to be evenly distributed across the protein
3-5 peptides per protein
3-5 transitions per peptide
Optimized MRM-MS assay
FIGURE 2
Absolute quantitation using MRM-MS in combination with isotope-labeled internal standard.
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