bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2023,
Номер
unknown
Опубликована: Ноя. 11, 2023
ABSTRACT
Liquid
chromatography-mass
spectrometry
(LC-MS)
based
top-down
proteomics
(TDP)
is
an
essential
method
for
the
analysis
of
intact
proteoforms.
The
accurate
quantification
individual
proteoforms
a
crucial
step
in
identifying
proteome-wide
alterations
different
biological
conditions.
Label-free
(LFQ)
most
common
proteoform
as
it
requires
no
additional
costly
labeling.
In
TDP,
due
to
frequent
co-elution
and
complex
signal
structures,
overlapping
signals
deriving
from
multiple
complicate
quantification.
Here,
we
introduce
FLASHQuant
MS1-level
LFQ
which
capable
automatically
resolving
quantifying
co-eluting
performs
highly
reproducible
short
runtimes
just
few
minutes
per
LC-MS
run.
To
validate
reported
by
FLASHQuant,
evaluated
them
with
identified
confirmed
tandem
mass
spectrometry,
showed
high
match
rates.
publicly
available
platform-independent
open-source
software
at
https://openms.org/flashquant/
.
Journal of Proteome Research,
Год журнала:
2023,
Номер
22(10), С. 3242 - 3253
Опубликована: Авг. 31, 2023
Proteome
profiles
of
precious
tissue
samples
have
great
clinical
potential
for
accelerating
disease
biomarker
discovery
and
promoting
novel
strategies
early
diagnosis
treatment.
However,
tiny
are
often
difficult
to
handle
analyze
with
conventional
proteomic
methods.
Automated
digital
microfluidic
(DMF)
workflows
facilitate
the
manipulation
size-limited
samples.
Here,
we
report
assessment
a
DMF
microproteomics
workflow
enabled
by
photocleavable
surfactant
analysis
minute
The
4-hexylphenylazosulfonate
(Azo)
was
found
fast
droplet
movement
on
enhance
proteomics
analysis.
Comparisons
Azo
n-Dodecyl
β-d-maltoside
(DDM)
using
small
HeLa
digest
standards
MCF-7
cell
digests
revealed
distinct
differences
at
peptide
level
despite
similar
results
protein
level.
applied
sample
preparation
∼3
μg
biopsies
from
murine
brain
tissue.
A
total
1969
proteins
were
identified
in
three
samples,
including
established
neural
biomarkers
related
synaptic
signaling.
Going
forward,
propose
that
Azo-enabled
has
advance
practical
application
Abstract
Low‐input
proteomics,
also
referred
to
as
micro‐
or
nanoproteomics,
has
become
increasingly
popular
it
allows
one
elucidate
molecular
processes
in
rare
biological
materials.
A
major
prerequisite
for
the
analytics
of
minute
protein
amounts,
e.g.,
derived
from
low
cell
numbers,
down
single
cells,
is
availability
efficient
sample
preparation
methods.
Digital
microfluidics
(DMF),
a
technology
allowing
handling
and
manipulation
liquid
volumes,
recently
been
shown
be
powerful
versatile
tool
address
challenges
low‐input
proteomics.
Here,
an
overview
provided
on
recent
advances
proteomics
using
DMF.
In
particular,
capability
DMF
isolate
proteomes
cells
small
model
organisms,
perform
all
necessary
chemical
steps,
such
denaturation
proteolytic
digestion
on‐chip,
are
highlighted.
Additionally,
prerequisites
making
these
steps
compatible
with
follow‐up
analytical
methods
chromatography‐mass
spectrometry
will
discussed.
Analytical Chemistry,
Год журнала:
2024,
Номер
96(43), С. 17227 - 17234
Опубликована: Окт. 18, 2024
Accurate
quantification
of
individual
proteoforms
is
a
crucial
step
in
identifying
proteome-wide
alterations
different
biological
conditions.
Intact
have
been
analyzed
predominantly
by
liquid
chromatography-mass
spectrometry
(LC-MS)-based
top-down
proteomics
(TDP)
and
quantified
primarily
the
label-free
(LFQ)
method,
as
it
requires
no
additional
costly
labeling.
In
TDP,
due
to
frequent
coelution
complex
signal
structures,
overlapping
signals
deriving
from
multiple
complicate
accurate
quantification.
Here,
we
introduce
FLASHQuant
for
MS1-level
LFQ
analysis
which
capable
automatically
resolving
quantifying
coeluting
proteoforms.
benchmark
tests
performed
with
both
spike-in
proteins
proteome-level
mixture
data
sets,
was
shown
perform
highly
reproducible
short
runtimes
just
few
minutes
per
LC-MS
run.
particular,
demonstrated
that
boosts
accuracy.
publicly
available
platform-independent
open-source
software
at
https://openms.org/flashquant/,
accompanied
simple
alignment
algorithm
ConsensusFeatureGroupDetector
runs.
Abstract
Top‐down
proteomics
(TDP)
directly
analyzes
intact
proteins
and
thus
provides
more
comprehensive
qualitative
quantitative
proteoform‐level
information
than
conventional
bottom‐up
(BUP)
that
relies
on
digested
peptides
protein
inference.
While
significant
advancements
have
been
made
in
TDP
sample
preparation,
separation,
instrumentation,
data
analysis,
reliable
reproducible
analysis
still
remains
one
of
the
major
bottlenecks
TDP.
A
key
step
for
robust
is
establishment
an
objective
estimation
false
discovery
rate
(FDR)
proteoform
identification.
The
most
widely
used
FDR
scheme
based
target‐decoy
approach
(TDA),
which
has
primarily
established
BUP.
We
present
evidence
TDA‐based
may
not
work
at
due
to
overlooked
factor,
namely
erroneous
deconvolution
precursor
masses,
leads
incorrect
estimation.
argue
identification
fact
protein‐level
rather
unless
error
taken
into
account.
To
address
this
issue,
we
propose
a
formula
correct
bias
by
combining
rate.
Microfluidic
systems
incorporating
magnetic
droplets
have
emerged
as
a
focal
point
of
significant
interest
within
the
biomedical
domain.
The
allure
these
lies
in
their
capacity
to
offer
precise
control,
enable
contactless
operation,
and
accommodate
minimal
sample
concentration
requirements.
Such
remarkable
features
serve
mitigate
errors
arising
from
human
operation
other
factors
during
cell
or
molecular
detection.
By
providing
innovative
solutions
for
diagnostics
immunoassay
applications,
droplet
microfluidics
enhance
accuracy
efficiency
procedures.
This
review
undertakes
comprehensive
examination
research
progress
microfluidic
centered
around
droplets.
It
adheres
sequential
presentation
approach,
commencing
fundamental
principles,
specifically
generation
on
chip,
proceeding
transmission
mixing
microchannel
Angewandte Chemie,
Год журнала:
2023,
Номер
135(45)
Опубликована: Сен. 1, 2023
Abstract
Mass
spectrometry
has
emerged
as
a
mainstream
technique
for
label‐free
proteomics.
However,
proteomic
coverage
trace
samples
is
constrained
by
adsorption
loss
during
repeated
elution
at
sample
pretreatment.
Here,
we
demonstrated
superparamagnetic
composite
nanoparticles
functionalized
with
molecular
glues
(MGs)
to
enrich
proteins
in
human
biofluid.
We
showed
high
protein
binding
(>95
%)
and
recovery
(≈90
rates
anchor‐nanoparticles.
further
proposed
Streamlined
Workflow
based
on
Anchor‐nanoparticles
Proteomics
(SWAP)
method
that
enabled
unbiased
capture,
digestion
pure
peptides
one
single
tube.
SWAP
quantify
over
2500
groups
100
HEK
293T
cells.
adopted
profile
proteomics
aqueous
humor
from
cataract
(
n
=15)
wet
age‐related
macular
degeneration
=8)
patients,
quantified
≈1400
5
μL
humor.
simplifies
preparation
steps,
minimizes
improves
previous
samples.
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2023,
Номер
unknown
Опубликована: Ноя. 11, 2023
ABSTRACT
Liquid
chromatography-mass
spectrometry
(LC-MS)
based
top-down
proteomics
(TDP)
is
an
essential
method
for
the
analysis
of
intact
proteoforms.
The
accurate
quantification
individual
proteoforms
a
crucial
step
in
identifying
proteome-wide
alterations
different
biological
conditions.
Label-free
(LFQ)
most
common
proteoform
as
it
requires
no
additional
costly
labeling.
In
TDP,
due
to
frequent
co-elution
and
complex
signal
structures,
overlapping
signals
deriving
from
multiple
complicate
quantification.
Here,
we
introduce
FLASHQuant
MS1-level
LFQ
which
capable
automatically
resolving
quantifying
co-eluting
performs
highly
reproducible
short
runtimes
just
few
minutes
per
LC-MS
run.
To
validate
reported
by
FLASHQuant,
evaluated
them
with
identified
confirmed
tandem
mass
spectrometry,
showed
high
match
rates.
publicly
available
platform-independent
open-source
software
at
https://openms.org/flashquant/
.
