Biology Reference
In-Depth Information
standards.
75,76
The SILAP standards are secre-
tome products
72
e
74,77,78
of SILAC human cell
cultures. Both SILAP and super SILAC stan-
dards have been demonstrated for quantitative
pro
options for assembling improved label-free
quantitative proteomic platforms.
92
e
94
In addi-
tion, there is an emerging practice which
uses both label-free and stable isotope-based
quantitation for proteomic samples that are
combined with isotope labeled counterparts or
standards.
95
ling
75,76,79
anddirecteddiscovery
72
e
74,80
of human biomarker candidates, with the
increasing acceptance from the community.
However, SILAC proteome mixtures can hardly
provide reference standards for analyzing all of
the proteins in human tissues and body
Protein Biomarker Discovery
Advances of the last decade in proteomic
analysis instrumentation and informatics have
allowed for rapid expansion of the potential
protein biomarker pool for disease diagnosis,
prognosis, and personalized therapeutics.
However, many novel protein biomarkers are
expected to have low abundances in sera or
plasma. It is a challenge for even the best
MS-based proteomic platform to accurately
and con
fluids.
Label-Free Quantitative Proteomics
Label-free quantitative proteomics
9,81,82
is
particularly attractive to discover potential
protein biomarkers in clinical samples. Without
added reference proteome samples carrying
stable isotope labels, the label-free methods
allow for the best LOD and LOQ afforded by
a particular MS-based platform. In addition,
sample preparations are relatively simple,
without tagging and the associated cleanup
steps. Recent advances in MS instrumentation
and in proteomic informatics further promote
the application of label-free approaches. In
general, label-free quantitation is achieved by
measurements of peptides for their MS signal
intensities
83,84
and/or spectral counts.
85,86
Label-free methods analyze individual samples
separately; therefore, there is no sample dilution
concern. However, sample throughput of label-
free proteomic quantitation is the lowest among
all quantitative proteomic technologies.
Thanks to the possibility of achieving intrinsic
LOQs available for a given mass spectrometer
and the simplicity of preparing samples for
label-free quantitation, this quantitation strategy
has been applied for the analyses of complex pro-
teome samples including plasma, sera,
9,87
dently measure the low-abundance
proteins in sera or plasma. Therefore it is crit-
ical to use tissue samples or body
fluids in/
close to the disease area in the stage of protein
biomarker discovery.
1
The discovery stage typi-
cally utilizes global quantitative pro
ling tech-
nologies that have relatively compromised
LOQs and LODs. Therefore, sampling pro-
teomes which are close to or at the disease loca-
tion can increase the concentration of disease-
relevant, low-abundance proteins and thus
increase the rate for new protein biomarker
identi
cation.
Differentially Expressed Proteins
MS-based quantitative pro
ling of tissue
samples is an attractive option for biomarker
discovery due to the large numbers of sample
libraries
cere-
available. One of
these
libraries
fluid,88
88
urine,
89,90
and tissues.
91
However, the complexity of proteome samples
demands for multiple-step separations, and
run-to-run variations in peptide elution time
impose unpredictable matrix effects for peptide
ionization. New generations of high resolution
and high accuracy MS instruments provide new
brospinal
is formalin-
n-embedded (FFPE) tis-
sues gathered post-mortem.
96
Laser capture
microdissection (LCM) has been used to further
dissect FFPE tissues to analyze only areas of
interest.
97
This technique is bene
xed paraf
cial when only
aparticularareaisofinterestortocompare
different segments in the same tissue.
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