Mass SpectrometryeBased Approach for Protein Biomarker Verification - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 399

well as issues encountered applying the approach
for peptide analysis will also be discussed.

isotope-labeled internal peptide standard spiked
in with known amount.
The targetedMS-based approach to be applied
to biomarker veri

cation
of peptides derived from the protein biomarker
candidates. Once the protein biomarker can-
didates to be veri

cation relies on quanti

MRM-MS ASSAY GENERATION FOR
PROTEIN QUANTITATION

ed are determined, the
peptides targeted for their accurate quantitation
have to be unique in the proteome to these
speci

Multiple-reaction monitoring mass spectrom-
etry (MRM-MS) is generally done using triple
quadrupole or hybrid triple quadrupole ion
trap mass spectrometers because of their short
duty cycle as well as their linear quantitation
range. MRM-MS is performed by isolating the
precursor ion in the

c proteins. Peptides and subsequent transi-
tions suitable for MRM-MS assays must be evalu-
ated and optimized to achieve best performance
with least interferences. In fact, peptides seen in
discovery stages are not necessarily the best can-
didates to give best performance in MRM-MS
assays. Additional optimization of MRM-MS
experiments are still required to narrow down
the best peptides and/or the best transitions to
monitor in the interested sample matrix to
achieve the best sensitivity and reproducibility.
The best way to generate optimized MRM-MS
assays obviously is to start with recombinant
proteins if they are available. However, in most
cases, the recombinant proteins are not available
and/or too expensive/dif

first quadrupole (Q1), frag-
menting it within Q2, and monitoring the
optimum fragment ions using Q3. The selectivity
and speci

city is achieved through a combina-
tion of Q1 isolating only precursor peptide ions
within a narrow mass window and monitoring
fragment

ion masses corresponding to the

speci c precursor ions in Q3. The selectivity typi-

cally increases when multiple transitions are
monitored for the same precursor ion, with
three to five transitions typically being used

for selectivity purpose. As shown in Figure 1 ,
absolute quantitation is achieved using a combi-
nation of stable isotope dilution (SID) and
MRM-MS by comparing the peak area obtained
from the endogenous peptide (best performing
transition peak area or peak area from combined
transitions) and that obtained from the stable

cult to generate. Pep-
tides observed during discovery stage and/or
selected from predicated high-responding
peptides after an in silico proteolytic digestion 13
after further filtering using certain criteria are

used to generate the MRM-MS assay. Besides
using recombinant proteins, crude synthetic

Protein

Q3

Q1 Q2

Area A

k

k

k

LC

digest

k

k k

Area B

k

k

k

k

Precursor isolation CID Fragment(s) isolation

Heavy stable isotope standard

FIGURE 1 MRM-MS method development and optimization workflow for protein quantitation.