Quantitative Proteomics in Development of Disease Protein Biomarkers - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 263

the post-acquisition data analysis. One type of
quantitation uses a selective ion transmission
via the

make it practically useful for analyzing complex
proteome samples.

first mass analyzer, and after the gas-
phase fragmentation of the selected precursor
ion, the fragment ions are analyzed by a second
mass analyzer of high resolution and high accu-
racy. The intensity of multiple fragment ions can
be added to improve LOQ. The intrinsic advan-
tage of high resolution, high accuracy of the frag-
ment ion analysis can also signi

ANALYTE MULTIPLEXING AND
SAMPLE THROUGHPUT

Capable of analyzing many ions of different
m/z ratios is intrinsic to MS. In the pipeline of
the biomarker development, this capability is
utilized for particular purposes of different
development stages ( Figure 3 ). In the early
biomarker candidate discovery stage, MS-based
quantitative proteomics largely uses the capa-
bility for multiplex analysis of peptides origi-
nated from hundreds and thousands of
proteins; this is particularly true for label-free
quantitative proteomics. For stable-isotope-
enabled quantitative proteomics, a small portion
of this capability is used for concurrent analysis
of several samples. As a result, this practice
increases the sample throughput for quantitative
proteomic pro

cantly improve

the speci city of targeted quantitation of signa-

ture peptides of protein biomarkers. The broad-
band mass analysis of fragment ions, compared
to conventional QqQ-based targeted quantita-
tion, also eliminates the need for the pre-
selection of the best transitions like that being
implemented in the MRM experiment design.
This facilitates the development of new
biomarkers. Storage of mass spectra allows for
post-acquisition data analysis, compared to the
fact

that there is no spectrum generated in

MRM MS.
Without selecting particular precursor ions of
signature peptides, the high resolution, high
accuracy mass analysis of fragment ions gener-
ated from all intact ions is also possible, leading
to the development of another type of

ling for biomarker candidates,
although the simultaneous analysis is mainly
meant to improve the quantitation accuracy
and precision. By the community convention,
this type of concurrent proteomic measurements
of several samples is also referred to as multi-
plexed analysis. This class of quantitative proteo-
mic analysis has been driving the technology
development of the

”
quantitation. For highly complex human pro-
teome samples, the high resolution, high accu-
racy mass determination is still challenging,
however, to deconvolute the highly complex
spectra generated from all coming-intact ions at
a given time. Experimental examination of the
speci

“ targeted

field and is now fruitful for
producing disease biomarker candidates with
ever-increasing con dence.

With the progress in the biomarker develop-
ment pipeline, the number of proteins of interest
decreases and the number of samples increases. 1
This change demands a shift in using this capa-
bility from analyzing large numbers of proteins
in several human samples to measuring several
to a few tens of proteins in large numbers of
samples ( Figure 3 ). 1,183 The latter analysis is in
the domain of targeted proteomics mainly using
various MRM MS methods.
The sample throughput presents a major tech-
nology bottleneck for MS-based biomarker

city and limits of this type of targeted
quantitation is being explored and related new
methods are being developed. Instrumentation
that allows for a medium level of precursor ion
selection is also being developed; peptide intact
ions within a few tens to a couple of hundreds
are broadly selected for gas-phase dissociation
and fragment ion analysis. Although the concept
and demonstrations for these types of parallel
fragmentation of multiple ions have been exist-
ing for some time, 179 e 182

it is only the newest

high-performance mass spectrometers that