Biology Reference
In-Depth Information
SRM assays are developed using either exper-
imental proteome identi
removed by immunodepletion using the af
nity
columns.
107,110
cation data or publicly
available databases such as Peptide Atlas
101
or
GPM proteome database.
102
Advantages of
these databases include integration of hundreds
of experiments and unique algorithms to rank
proteotypic peptides by their performance in
LC-MS/MS experiments and to predict peptides
suitable for SRM assay development. Synthetic
peptides can also be used at this point to facili-
tate assay development. Software tools designed
to aid in SRM assay development include
commercial software provided by instrument
vendors, such as Pinpoint
(Thermo Fisher
Inc.) and MRMPilot
(AB Sciex Inc.), as
well as license-free Skyline,
103
MRMaid,
104
mProphet,
105
and SRMCollider.
106
Among all
MS techniques, SRM assays remain the methods
of
Alternatively,
low-abundance
proteins can be enriched by af
cation
using antibodies or aptamers
111
e
113
; however,
this approach has a reduced multiplexing poten-
tial. Similar approaches, such as SISCAPA,
employ antibodies developed against proteo-
typic peptides.
114,115
Because antibody develop-
ment against synthetic peptides is more
straightforward relative to intact proteins, use
of such approaches is increasing. Improved sensi-
tivity (down to 1 ng/mL) and increased multi-
plexing
nity puri
and
throughput
capabilities
of
SISCAPA assays enable accurate veri
cation of
biomarker candidates in blood plasma.
115,116
In
addition, as many known protein biomarkers
in clinical use are post-translationally modi-
fied with N-glycosylation,
117
nity
chromatography is sometimes used to enrich
N-glycoproteins and N-glycopeptides prior to
LC-SRM analysis.
96,117
lectin af
choice
for protein quanti
cation and
biomarker veri
cation due to their sensitivity,
high-throughput capabilities, and multiplexing
potential.
B
IOMARKER VERIFICATIO
N
Separation and Enrichment Strategies
for Quanti
cation of Low-Abundance
cation
phase, anywhere from dozens to hundreds of
proteins are usually selected as potential
biomarkers. Large variation of analysis and
poor reproducibility of commonly used label-
free approaches constitute serious technological
limitations of the identi
Upon completion of the protein identi
Proteins
Relatively low sensitivity and moderate
throughput of mass spectrometry-based protein
assays (
100 ng/mL) remain two major limita-
tions of their use for biomarker validation
studies and clinical analysis. Because blood
serum levels of many established clinical
biomarkers are in the 10 pg/mL to 10 ng/mL
range,
18
high-abundance proteins mask low-
abundance
w
cation phase. Biological
factors such as intraindividual variations of
protein levels during the day as well as wide
interindividual distribution of physiological
levels of proteins in healthy individuals also
result
biomarkers
and
signi
cantly
compromise their quanti
cation by mass spec-
trometry. Thus, LC-SRM measurement of low-
abundance proteins can be achieved only
through additional separation and enrichment.
A set of strategies, such as strong anion- or
cation-exchange chromatography and isoelectric
focusing, are used to remove high-abundance
or enrich low-abundance proteins.
107
e
109
Major high-abundance proteins can also be
cant bias. The potential of
a certain protein biomarker should be con
in a signi
rmed
cation in the independent set of
samples. Even though there is a rapidly
increasing number of publications reporting iden-
ti
first by veri
cation of potential biomarkers, the rate of
newly approved protein biomarkers is steadily
decreasing in the last decade.
18,118
This decrease
can be partially explained by a high number of
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