Quantitative Proteomics in Development of Disease Protein Biomarkers - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 260

FIGURE 2 Multiple reaction monitoring MS on triple quadrupole mass spectrometer.

as an ion chromatogram for the precursor-
fragment ion pair. The corresponding m/z pairs
are called gas-phase transitions in MRM MS
experiments; they are predetermined during
the experiment design stage. It is also important
that new QqQ mass spectrometers be capable of
fast analysis of each ion pair; they can then
analyze the increased number of peptides in
a unit time, thus increasing the multiplexing
potential or sample throughput potential. 133
These further increase when high-performance
liquid chromatography (HPLC) is used as the
front-end separation method. 134
Selective ion transmission affords MRM MS
analysis with great LOQ, high speci

peptides and their counterpart labeled reference;
it also quickly reduces the sample complexity of
plasma and sera digests, 139 e 142 making possible
the fast liquid chromatography-stable isotope-
dilution (LC-SID)-MRM-MS quantitation with
minimal matrix interference. At the peptide level,
human proteome digests can also be simpli

ed
by the selective sampling of signature peptides
of protein biomarkers; examples include the
application of the ICAT technology, sampling of
protein terminal peptides, 143 e 148 and selective
enrichment of peptides with post-translational
modi cations. 149

Enrichment of protein biomarkers through
pretreatment of plasma and sera samples can
also signi

city, and
a wide dynamic range. The characteristics
increase further when human samples are sub-
jected to prior preparation steps. For human pro-
teome samples such as plasma and sera,
preparation techniques include high-abundant
protein depletion, prefractionation, and target
analyte enrichment. 135,136 In a human plasma
sample, 99% of the total protein content consists
of albumin, haptoglobin, hemopexin, immuno-
globulins, and other high-abundance species.
Separation and enrichment strategies have been
developed to alleviate the biological matrix
effect. These strategies include stable isotope
standards and capture by antipeptide antibodies
(SISCAPA), 137,138 combined fractional diagonal
chromatography (COFRADIC), immuno-
depletion, peptide fractionation, and multi-
dimensional protein identi

cantly improve the sample loading
capacity, LOQ, and the linear dynamic range for
targeted quantitation of the proteins. Depletion
of high-abundance proteins in human plasma
and sera using af

cantly
increase the sample loading capacity for medium-
and low-abundance proteins. The caveat of deple-
tion, however,

nity materials can signi

is the loss of low-abundance

proteins that have signi nity to the

high-abundance proteins. Accordingly, strategies
have been developed to use the combination of
large sample loading and multiple-dimensional
separation for targeted quantitation of low-
abundance protein biomarkers without major
protein depletion. 150,151

cant af

Signature Peptides for Protein Targets
For a given protein, selection of a signature
peptide and optimum transitions for the peptide
in MRM analysis is crucial. A number of criteria

cation technique
(MuDPIT). The SISCAPA technology uses anti-
peptide antibodies to speci cally enrich signature