Additional File 2, Comparison of different proteomics-based techniques
METHOD
DESCRIPTION






ADVANTAGES
DISADVANTAGES
SENSITIVITY
2D GEL ELECTROPHORESIS/MASS SPECTROMETRY
Separation
of  Ability to identify  Proteins expressed at  Detection
complex
proteins
unknown proteins
low abundance may be
sensitivity is in the
via
2D
gel  Detects
protein
missed
nanogram range
electrophoresis
modification
 Unsuited
for
(50 ng/spot for
based charge and
(phosphorylation
diagnostic application
Coomassie Blue;
size
and methylation)
 Limited
1 ng/spot for silver
Major
protein  Used for various
reproducibility
and
stain)
identification by MS
biological samples,
high rate of false  Using fluorescent
Detects about 2000including
tissue,
identification
2D-differential gel
2500 spots/gel
blood and other  Limited
dynamic
electrophoresis
biological fluids
range
(2D-DIGE),
 semi-quantitative
sensitivity
improves by 10
fold (CyDye label)
LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY
LC
to
separate  Ability to identify  Proteins expressed at  Detection
proteins in a sample,
unknown proteins
low abundance may be
sensitivity is in the
with sequential LC  improved
missed
nanogram range
for
improved
separation
 Unsuited
for
or ~20 cells
separation efficiency
efficiency
diagnostic application  1% false positive
MS
to
compared to 2D gel  Limited
rate
systematically
 Used for various
reproducibility
and
identify the major
biological samples,
high rate of false
proteins
including
tissue,
identification
Detects over 1000
blood and other  Limited
dynamic
proteins/run
biological fluids
range
 semi-quantitative
PROTEIN ARRAY
 Individual protein
immobilization on a
solid-support (glass
or membrane)
 Individual proteins
identified by labeled
antibodies
 Detects over 1000
proteins/array
 Multiple whole-cell
or tissue lysate
immobilization on
individual spots on a
solid
support
(similar to tissue
microarray format)
 Presence of specific
proteins are detected
by antibody
 Detects
<
100
proteins/array
 High sensitivity and  Limited
protein
specificity
availability
from
 Good quantitation
complex
protein
range
production
process
 High
(expression
and
throughput/density
purification)
amenable
for  Limited access to a
automation
large
number
of
 Economical
and
affinity antibodies for
low
sample
detection.
consumption
 Lots of data from
single experiment
 Software
and
hardware tools may
be shared with
DNA microarray
REVERSE PHASE PROTEIN ARRAY
 Highly
sensitive  Detection sensitivity
detection
of
may be compromised
proteins
from
loss
native
 High throughput,
protein conformation
i.e. a large number
when surface spotted
of samples on one  Limited sensitivity to
slide
detect low abundance
 Minimal
sample
proteins
required
 Specificity may be
 Reduced number of
compromised
from
antibodies needed
non-specific antibody
to detect protein
binding (i.e. potential
for high background)
 Limited number of
available
signaling
protein-specific
antibodies
ANTIBODY ARRAY
 Detection
sensitivity is in the
ng/ml range
 Detection
sensitivity is in the
picogram range
 Increased
sensitivity
 Using
laser
capture
microdissection,
lysates can be
analyzed with as
few as 10 cells
 Capture antibodies
are spotted and fixed
on a solid surface
 Proteins (antigens)
are captured on the
array surface and
detected by a second
antibody specific for
a different epitopes
than
capture
antibody (sandwich
format)
 Detects
<
100
proteins/array
 Complex proteins in
a sample (cells or
tissue) are separated
via
gel
electrophoresis
 Proteins
then
transfers
to
nitrocellular
membrane
 Proteins detected by
multichannel
immunoblot (similar
to Western Blot)
 Detects up to 300
proteins/run
 Highly
specific  Protein
complexity  Detection
from dual antibody
and denaturation may
sensitivity is in the
detection
affect
antigenlow pg/ml range
 Highly sensitive
antibody interaction
 High
throughput  Need for high-affinity
and amenable for
and specific antibodies
automation
for
capture
and
 Possible to detect
detection
protein
 Limited
dynamic
modifications
range of 2 or 3 orders
(phosphorylation,
of magnitude
methylation, etc) by
modificationspecific antibodies
 Suitable for clinical
applications
PATHWAY ARRAY
 Highly
sensitive  Limited availability of  Detection limit of
with detection of
signaling-related
1 ng for each
low
abundance
antibodies
protein
with
proteins
 Relative low throughchemiluminescenc
 Highly specific (as
put (one sample per
e; 0.1 ng with
determined
by
gel
fluorescence
immunoreactivity
 Limited
dynamic  Linear detection
and size)
range of 2 or 3 orders
range is 100 fold
 High accuracy and
of magnitude
for ECL and 1,000
reproducibility
for fluorescence.
 Minimal antibody
required for each
sample
 Detects
protein
modifications
(phosphorylation,
methylation, etc)
BEAD-BASED ARRAY
 Either
capture  Highly
sensitive  Protein
complexity  Detection limit is
antibody or proteins
and specific
and
denaturation
sufficient
to
are coated on beads  High
throughput
affecting
antigencapture
low
 Detection
of
and amenable for
antibody interaction
abundance protein
proteins by labeled
automation
 Need for high-affinity
analytes down to
antibodies (similar  Detects
protein
and specific antibodies
the pg/mL range
to antibody array or
modifications
for
capture
and
ELISA)
(phosphorylation,
detection
 Detects
50-100
methylation, etc) by  Limited
dynamic
proteins/run
modification
range of 2 or 3 orders
specific antibodies
of magnitude
 Suitable for clinical
applications
METHOD
DESCRIPTION






ADVANTAGES
DISADVANTAGES
SENSITIVITY
2D GEL ELECTROPHORESIS/MASS SPECTROMETRY
Separation
of  Ability to identify  Proteins expressed at  Detection
complex
proteins
unknown proteins
low abundance may be
sensitivity is in the
via
2D
gel  Detects
protein
missed
nanogram range
electrophoresis
modification
 Unsuited
for
(50 ng/spot for
based charge and
(phosphorylation
diagnostic application
Coomassie Blue;
size
and methylation)
 Limited
1 ng/spot for silver
Major
protein  Used for various
reproducibility
and
stain)
identification by MS
biological samples,
high rate of false  Using fluorescent
Detects about 2000including
tissue,
identification
2D-differential gel
2500 spots/gel
blood and other  Limited
dynamic
electrophoresis
biological fluids
range
(2D-DIGE),
 semi-quantitative
sensitivity
improves by 10
fold (CyDye label)
LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY
LC
to
separate  Ability to identify  Proteins expressed at  Detection
proteins in a sample,
unknown proteins
low abundance may be
sensitivity is in the
with sequential LC  improved
missed
nanogram range
for
improved
separation
 Unsuited
for
or ~20 cells
separation efficiency
efficiency
diagnostic application  1% false positive
MS
to
compared to 2D gel  Limited
rate
systematically
 Used for various
reproducibility
and
identify the major
biological samples,
high rate of false
proteins
including
tissue,
identification
Detects over 1000
blood and other  Limited
dynamic
proteins/run
biological fluids
range
 semi-quantitative
PROTEIN ARRAY
 Individual protein
immobilization on a
solid-support (glass
or membrane)
 Individual proteins
identified by labeled
antibodies
 Detects over 1000
proteins/array
 Multiple whole-cell
or tissue lysate
immobilization on
individual spots on a
solid
support
(similar to tissue
microarray format)
 Presence of specific
proteins are detected
by antibody
 Detects
<
100
proteins/array
 High sensitivity and  Limited
protein
specificity
availability
from
 Good quantitation
complex
protein
range
production
process
 High
(expression
and
throughput/density
purification)
amenable
for  Limited access to a
automation
large
number
of
 Economical
and
affinity antibodies for
low
sample
detection.
consumption
 Lots of data from
single experiment
 Software
and
hardware tools may
be shared with
DNA microarray
REVERSE PHASE PROTEIN ARRAY
 Highly
sensitive  Detection sensitivity
detection
of
may be compromised
proteins
from
loss
native
 High throughput,
protein conformation
i.e. a large number
when surface spotted
of samples on one  Limited sensitivity to
slide
detect low abundance
 Minimal
sample
proteins
required
 Specificity may be
 Reduced number of
compromised
from
antibodies needed
non-specific antibody
to detect protein
binding (i.e. potential
for high background)
 Limited number of
available
signaling
protein-specific
antibodies
ANTIBODY ARRAY
 Detection
sensitivity is in the
ng/ml range
 Detection
sensitivity is in the
picogram range
 Increased
sensitivity
 Using
laser
capture
microdissection,
lysates can be
analyzed with as
few as 10 cells
 Capture antibodies
are spotted and fixed
on a solid surface
 Proteins (antigens)
are captured on the
array surface and
detected by a second
antibody specific for
a different epitopes
than
capture
antibody (sandwich
format)
 Detects
<
100
proteins/array
 Complex proteins in
a sample (cells or
tissue) are separated
via
gel
electrophoresis
 Proteins
then
transfers
to
nitrocellular
membrane
 Proteins detected by
multichannel
immunoblot (similar
to Western Blot)
 Detects up to 300
proteins/run
 Highly
specific  Protein
complexity  Detection
from dual antibody
and denaturation may
sensitivity is in the
detection
affect
antigenlow pg/ml range
 Highly sensitive
antibody interaction
 High
throughput  Need for high-affinity
and amenable for
and specific antibodies
automation
for
capture
and
 Possible to detect
detection
protein
 Limited
dynamic
modifications
ranges of 2 or 3 orders
(phosphorylation,
of magnitude
methylation, etc) by
modificationspecific antibodies
 Suitable for clinical
applications
PATHWAY ARRAY
 Highly
sensitive  Limited availability of  Detection limit of
with detection of
signaling-related
1 ng for each
low
abundance
antibodies
protein
with
proteins
 Relative low throughchemiluminescenc
 Highly specific (as
put (one sample per
e; 0.1 ng with
determined
by
gel
fluorescence
immunoreactivity
 Limited
dynamic  Linear detection
and size)
ranges of 2 or 3 orders
range is 100 fold
 High accuracy and
of magnitude
for ECL and 1,000
reproducibility
for fluorescence.
 Minimal antibody
required for each
sample
 Detects
protein
modifications
(phosphorylation,
methylation, etc)
BEAD-BASED ARRAY
 Either
capture  Highly
sensitive  Protein
complexity  Detection limit is
antibody or proteins
and specific
and
denaturation
sufficient
to
are coated on beads  High
throughput
affecting
antigencapture
low
 Detection
of
and amenable for
antibody interaction
abundance protein
proteins by labeled
automation
 Need for high-affinity
analytes down to
antibodies (similar  Detects
protein
and specific antibodies
the pg/mL range
to antibody array or
modifications
for
capture
and
ELISA)
(phosphorylation,
detection
 Detects
50-100
methylation, etc) by  Limited
dynamic
proteins/run
modification
ranges of 2 or 3 orders
specific antibodies
of magnitude
 Suitable for clinical
applications