bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2019,
Номер
unknown
Опубликована: Июнь 9, 2019
Abstract
Macrophages
are
innate
immune
cells
with
diverse
functional
and
molecular
phenotypes.
This
diversity
is
largely
unexplored
at
the
level
of
single-cell
proteomes
because
limitations
quantitative
protein
analysis.
To
overcome
this
limitation,
we
developed
SCoPE2,
which
substantially
increases
accuracy
throughput
while
lowering
cost
hands-on
time
by
introducing
automated
miniaturized
sample
preparation.
These
advances
enable
us
to
analyze
emergence
cellular
heterogeneity
as
homogeneous
monocytes
differentiate
into
macrophage-like
in
absence
polarizing
cytokines.
SCoPE2
quantified
over
3,042
proteins
1,490
single
macrophages
ten
days
instrument
time,
allow
discern
cell
type.
Furthermore,
data
uncover
a
continuous
gradient
proteome
states
for
macrophages,
suggesting
that
macrophage
may
emerge
correlates
inflammatory
axis
classically
alternatively
activated
macrophages.
Parallel
measurements
transcripts
10x
Genomics
suggest
our
20-fold
more
copies
than
RNA
per
gene,
thus
supports
quantification
improved
count
statistics.
The
joint
distributions
allowed
exploring
regulatory
interactions,
such
between
tumor
suppressor
p53,
its
transcript,
genes
regulated
p53.
Our
methodology
lays
foundation
analysis
mass-spectrometry
demonstrates
potential
inferring
transcriptional
post-transcriptional
regulation
from
variability
across
cells.
Figure
Abstract
Background
Macrophages
are
innate
immune
cells
with
diverse
functional
and
molecular
phenotypes.
This
diversity
is
largely
unexplored
at
the
level
of
single-cell
proteomes
because
limitations
quantitative
protein
analysis.
Results
To
overcome
this
limitation,
we
develop
SCoPE2,
which
substantially
increases
accuracy
throughput
while
lowering
cost
hands-on
time
by
introducing
automated
miniaturized
sample
preparation.
These
advances
enable
us
to
analyze
emergence
cellular
heterogeneity
as
homogeneous
monocytes
differentiate
into
macrophage-like
in
absence
polarizing
cytokines.
SCoPE2
quantifies
over
3042
proteins
1490
single
macrophages
10
days
instrument
time,
quantified
allow
discern
cell
type.
Furthermore,
data
uncover
a
continuous
gradient
proteome
states
for
macrophages,
suggesting
that
macrophage
may
emerge
Parallel
measurements
transcripts
10×
Genomics
suggest
our
20-fold
more
copies
than
RNA
per
gene,
thus,
supports
quantification
improved
count
statistics.
allowed
exploring
regulatory
interactions,
such
interactions
between
tumor
suppressor
p53,
its
transcript,
genes
regulated
p53.
Conclusions
Even
environment,
heterogeneous.
correlates
inflammatory
axis
classically
alternatively
activated
macrophages.
Our
methodology
lays
foundation
analysis
mass
spectrometry
demonstrates
potential
inferring
transcriptional
post-transcriptional
regulation
from
variability
across
cells.
Molecular & Cellular Proteomics,
Год журнала:
2020,
Номер
19(11), С. 1739 - 1748
Опубликована: Авг. 26, 2020
MS-based
proteome
profiling
has
become
increasingly
comprehensive
and
quantitative,
yet
a
persistent
shortcoming
been
the
relatively
large
samples
required
to
achieve
an
in-depth
measurement.
Such
bulk
samples,
typically
comprising
thousands
of
cells
or
more,
provide
population
average
obscure
important
cellular
heterogeneity.
Single-cell
proteomics
capabilities
have
potential
transform
biomedical
research
enable
understanding
biological
systems
with
new
level
granularity.
Recent
advances
in
sample
processing,
separations
MS
instrumentation
now
make
it
possible
quantify
>1000
proteins
from
individual
mammalian
cells,
coverage
that
input
just
few
years
ago.
This
review
discusses
factors
parameters
should
be
optimized
across
workflow
for
single-cell
other
low-input
measurements.
It
also
highlights
recent
developments
advanced
field
opportunities
further
development.
Nature Communications,
Год журнала:
2021,
Номер
12(1)
Опубликована: Июнь 7, 2021
Large-scale
single-cell
analyses
are
of
fundamental
importance
in
order
to
capture
biological
heterogeneity
within
complex
cell
systems,
but
have
largely
been
limited
RNA-based
technologies.
Here
we
present
a
comprehensive
benchmarked
experimental
and
computational
workflow,
which
establishes
global
mass
spectrometry-based
proteomics
as
tool
for
large-scale
analyses.
By
exploiting
primary
leukemia
model
system,
demonstrate
both
through
pre-enrichment
populations
non-enriched
unbiased
approach
that
our
workflow
enables
the
exploration
cellular
this
aberrant
developmental
hierarchy.
Our
is
capable
consistently
quantifying
~1000
proteins
per
across
thousands
individual
cells
using
instrument
time.
Furthermore,
develop
(SCeptre)
effectively
normalizes
data,
integrates
available
FACS
data
facilitates
downstream
analysis.
The
presented
here
lays
foundation
implementing
studies
world.
Journal of the American Society for Mass Spectrometry,
Год журнала:
2021,
Номер
32(4), С. 872 - 894
Опубликована: Март 3, 2021
Biological
systems
are
composed
of
heterogeneous
populations
cells
that
intercommunicate
to
form
a
functional
living
tissue.
function
varies
greatly
across
cells,
as
each
single
cell
has
unique
transcriptome,
proteome,
and
metabolome
translates
differences
within
species
kingdoms.
Over
the
past
decade,
substantial
advancements
in
our
ability
characterize
omic
profiles
on
level
have
occurred,
including
multiple
spectroscopic
mass
spectrometry
(MS)-based
techniques.
Of
these
technologies,
spatially
resolved
approaches,
imaging
(MSI),
shown
most
progress
for
proteomics
metabolomics.
For
example,
reporter-based
methods
using
heavy
metal
tags
allowed
targeted
MS
investigation
proteome
at
subcellular
level,
development
technologies
such
laser
ablation
electrospray
ionization
(LAESI-MS)
now
mean
dynamic
metabolomics
can
be
performed
situ.
In
this
Perspective,
we
showcase
spatial
over
decade
highlight
important
aspects
related
high-throughput
screening,
data
analysis,
more
which
vital
success
achieving
proteomic
metabolomic
profiling
scale.
Finally,
broad
literature
summary,
provide
perspective
how
next
may
unfold
area
MS-based
Analytical Chemistry,
Год журнала:
2020,
Номер
92(15), С. 10588 - 10596
Опубликована: Июль 8, 2020
Single-cell
proteomics
can
provide
critical
biological
insight
into
the
cellular
heterogeneity
that
is
masked
by
bulk-scale
analysis.
We
have
developed
a
nanoPOTS
(nanodroplet
processing
in
one
pot
for
trace
samples)
platform
and
demonstrated
its
broad
applicability
single-cell
proteomics.
However,
because
of
nanoliter-scale
sample
volumes,
not
compatible
with
automated
LC-MS
systems,
which
significantly
limits
throughput
robustness.
To
address
this
challenge,
we
autosampler
allowing
fully
injection
from
nanowells
to
systems.
also
drying,
extraction,
loading
workflow
enable
reproducible
reliable
injection.
The
sequential
analysis
20
samples
containing
10
ng
tryptic
peptides
high
reproducibility
correlation
coefficients
>0.995
between
any
two
samples.
9.6,
16,
24
single
cells
per
day
using
120,
60,
30
min
LC
gradients,
respectively.
As
demonstration
proteomics,
was
first
applied
profiling
protein
expression
MCF10A
label-free
approach.
At
day,
an
average
256
proteins
identified
each
cell
number
increased
731
when
Match
Between
Runs
algorithm
MaxQuant
used.
Using
multiplexed
isobaric
labeling
approach
(TMT-11plex),
∼77
could
be
analyzed
day.
152
three
acute
myeloid
leukemia
lines,
resulting
total
2558
1465
quantifiable
(70%
valid
values)
across
cells.
These
data
showed
quantitative
cluster
distinct
groups
reveal
functionally
differences.
