Molecular & Cellular Proteomics,
Год журнала:
2024,
Номер
unknown, С. 100880 - 100880
Опубликована: Ноя. 1, 2024
Mass
spectrometry-based
proteome
profiling
of
trace
analytes
including
single
cells
benefits
from
liquid
chromatography
separations
operated
at
low
flow
rates
(e.g.,
<50
nL/min).
However,
high-pressure
binary
pumps
needed
to
achieve
such
are
not
commercially
available,
and
instead
require
splitting
the
gradient
low-nanoliter-per-minute
rates.
Gradient
can
waste
solvent
lead
inconsistencies.
To
address
this,
we
have
developed
a
method
for
creating
gradients
by
combining
plugs
mobile
phase
increasing
strength
together
in
column,
then
relying
on
Taylor
dispersion
form
desired
smooth
profile.
Additionally,
our
dramatically
reduces
costs,
as
only
isocratic
pump
is
required.
Following
development
profiles
both
10-
20-min
active
gradients,
measured
200
pg
injections
HeLa
digest
using
timsTOF
mass
spectrometer.
Finally,
investigated
differences
protein
expression
between
originating
two
different
colonies
ATG-knockout
cells.
Thousands
proteins
were
quantified,
potential
mechanism
explaining
differential
immune
responses
these
upon
exposure
viral
DNA
treatment
was
determined.
ABSTRACT
Single‐cell
proteomics
(SCP)
has
advanced
significantly
in
recent
years,
with
new
tools
specifically
designed
for
the
preparation
and
analysis
of
single
cells
now
commercially
available
to
researchers.
The
field
is
sufficiently
mature
be
broadly
accessible
any
lab
capable
isolating
performing
bulk‐scale
proteomic
analyses.
In
this
review,
we
highlight
work
SCP
that
lowered
barrier
entry,
thus
providing
a
practical
guide
those
who
are
newly
entering
field.
We
outline
fundamental
principles
report
multiple
paths
accomplish
key
steps
successful
experiment
including
sample
preparation,
separation,
mass
spectrometry
data
acquisition
analysis.
recommend
researchers
start
label‐free
workflow,
as
achieving
high‐quality
quantitatively
accurate
results
more
straightforward
than
label‐based
multiplexed
strategies.
By
leveraging
these
means,
can
confidently
perform
experiments
make
meaningful
discoveries
at
single‐cell
level.
Analytical Chemistry,
Год журнала:
2024,
Номер
96(28), С. 11439 - 11447
Опубликована: Июль 5, 2024
In
this
work,
we
describe
the
construction
and
application
of
a
repurposed
3D-printer
as
fraction
collector.
We
utilize
nano-LC
to
ensure
minimal
volumes
surfaces
although
any
LC
can
be
coupled.
The
setup
operates
high-pH
fractionation
system
capable
effectively
working
with
nanogram
scales
lysate
digests.
2D
RP–RP
demonstrated
superior
proteome
coverage
over
single-shot
data-dependent
acquisition
(DDA)
analysis
using
only
5
ng
human
cell
digest
performance
increasing
amounts
material.
found
that
allowed
60%
signal
recovery
at
peptide
level
and,
more
importantly,
observed
improved
protein
intensity
coverage,
which
indicates
complexity
reduction
afforded
by
outweighs
sample
losses
endured.
data-independent
(DIA)
wide
window
(WWA)
fractionated
samples
nearly
8000
proteins
identified
from
50
utility
was
further
investigated
for
phosphoproteomics
(>21
000
phosphosites
μg
starting
material)
pull-down
type
experiments
showed
substantial
improvements
experiments.
show
is
highly
versatile
powerful
tool
many
proteomics
workflows.
Expert Review of Proteomics,
Год журнала:
2024,
Номер
21(7-8), С. 271 - 280
Опубликована: Авг. 2, 2024
Introduction
Metaproteomics
offers
insights
into
the
function
of
complex
microbial
communities
while
it
is
also
capable
revealing
microbe-microbe
and
host-microbe
interactions.
Data-independent
acquisition
(DIA)
mass
spectrometry
an
emerging
technology,
which
holds
great
potential
to
achieve
deep
accurate
metaproteomics
with
higher
reproducibility
yet
still
facing
a
series
challenges
due
inherent
complexity
DIA
data.
Molecular & Cellular Proteomics,
Год журнала:
2024,
Номер
unknown, С. 100880 - 100880
Опубликована: Ноя. 1, 2024
Mass
spectrometry-based
proteome
profiling
of
trace
analytes
including
single
cells
benefits
from
liquid
chromatography
separations
operated
at
low
flow
rates
(e.g.,
<50
nL/min).
However,
high-pressure
binary
pumps
needed
to
achieve
such
are
not
commercially
available,
and
instead
require
splitting
the
gradient
low-nanoliter-per-minute
rates.
Gradient
can
waste
solvent
lead
inconsistencies.
To
address
this,
we
have
developed
a
method
for
creating
gradients
by
combining
plugs
mobile
phase
increasing
strength
together
in
column,
then
relying
on
Taylor
dispersion
form
desired
smooth
profile.
Additionally,
our
dramatically
reduces
costs,
as
only
isocratic
pump
is
required.
Following
development
profiles
both
10-
20-min
active
gradients,
measured
200
pg
injections
HeLa
digest
using
timsTOF
mass
spectrometer.
Finally,
investigated
differences
protein
expression
between
originating
two
different
colonies
ATG-knockout
cells.
Thousands
proteins
were
quantified,
potential
mechanism
explaining
differential
immune
responses
these
upon
exposure
viral
DNA
treatment
was
determined.
