Journal of Proteome Research,
Год журнала:
2024,
Номер
unknown
Опубликована: Дек. 2, 2024
Quantitative
analysis
of
proteins
and
their
post-translational
modifications
(PTMs)
in
complex
biological
samples
is
critical
to
understanding
cellular
biology
as
well
disease
detection
treatment.
Top-down
proteomics
methods
provide
a
"bird's
eye"
view
the
proteome
by
directly
detecting
quantifying
intact
proteoforms.
Here,
we
developed
high-throughput
quantitative
top-down
platform
probe
proteoform
phosphoproteoform
abundance
changes
HeLa
cells
result
treatment
with
staurosporine
(STS),
broad-spectrum
kinase
inhibitor.
In
total,
identified
quantified
1187
proteoforms
from
215
families.
Among
them,
55
37
families
were
significantly
changed
upon
STS
These
primarily
related
catabolic,
metabolic,
apoptotic
pathways
that
are
expected
be
impacted
inhibition.
addition,
manually
evaluated
25
expressed
one
or
more
phosphorylated
We
observed
same
family,
such
eukaryotic
initiation
factor
4E
binding
protein
1
(4EBP1),
differentially
regulated
relative
unphosphorylated
Combining
profiling
within
these
individual
results
comprehensive
picture
treatment-induced
cannot
achieved
using
bottom-up
methods.
Abstract
Top-down
proteomics
using
mass
spectrometry
facilitates
the
identification
of
intact
proteoforms,
that
is,
all
molecular
forms
proteins.
Multiple
past
advances
have
lead
to
development
numerous
sample
preparation
workflows.
Here
we
systematically
investigated
influence
different
steps
on
proteoform
and
protein
identifications,
including
cell
lysis,
reduction
alkylation,
enrichment,
purification
fractionation.
We
found
in
subset
proteoforms
identified
(for
example,
their
number,
confidence,
physicochemical
properties
artificially
generated
modifications).
The
various
strategies
resulted
complementary
substantially
increasing
proteome
coverage.
Overall,
13,975
from
2,720
proteins
human
Caco-2
cells.
results
presented
can
serve
as
suggestions
for
designing
adapting
top-down
particular
research
questions.
Moreover,
expect
sampling
bias
modifications
at
level
will
also
be
useful
improving
bottom-up
approaches.
Analytical Science Advances,
Год журнала:
2023,
Номер
4(5-6), С. 181 - 203
Опубликована: Май 29, 2023
Abstract
Top–down
proteomics
(TDP)
identifies,
quantifies,
and
characterises
proteins
at
the
intact
proteoform
level
in
complex
biological
samples
to
understand
function
cellular
mechanisms.
However,
analysing
using
TDP
is
still
challenging
due
high
sample
complexity
wide
dynamic
range.
High‐resolution
separation
methods
are
often
applied
prior
mass
spectrometry
(MS)
analysis
decrease
increase
throughput.
These
methods,
however,
may
not
be
efficient
enough
characterise
low
abundance
proteoforms
samples.
As
such,
multidimensional
techniques
(combination
of
two
or
more
with
orthogonality)
have
been
developed
that
demonstrate
improved
resolution
comprehensive
identification
TDP.
A
suite
couple
various
types
liquid
chromatography
(LC),
capillary
electrophoresis
(CE),
and/or
gel
electrophoresis‐based
approaches
analyse
Here,
we
reviewed
strategies
employed
for
TDP,
summarised
current
applications,
discussed
gaps
addressed
future.
Journal of Proteome Research,
Год журнала:
2025,
Номер
24(2), С. 850 - 860
Опубликована: Янв. 22, 2025
Precise
prefractionation
of
proteome
samples
is
a
potent
method
for
realizing
in-depth
analysis
in
top-down
proteomics.
PEPPI-MS
(Passively
Eluting
Proteins
from
Polyacrylamide
gels
as
Intact
species
MS),
gel-based
sample
fractionation
method,
enables
high-resolution
based
on
molecular
weight
by
highly
efficient
extraction
proteins
polyacrylamide
after
SDS-PAGE
separation.
Thereafter
it
essential
to
effectively
remove
contaminants
such
CBB
and
SDS
the
PEPPI
fraction
prior
mass
spectrometry.
In
this
study,
we
developed
complete,
robust,
simple
preparation
workflow
named
PEPPI-SP3
proteomics
combining
with
magnetic
bead-based
protein
purification
approach
used
SP3
(single-pot,
solid-phase-enhanced
preparation),
now
one
standard
methods
bottom-up
PEPPI-SP3,
extracted
gel
are
collected
surface
beads,
washed
organic
solvents,
recovered
intact
100
mM
ammonium
bicarbonate
containing
0.05%
(w/v)
SDS.
The
subjected
spectrometry
additional
using
an
anion-exchange
StageTip.
Performance
validation
human
cell
lysates
showed
significant
improvement
low-molecular-weight
recovery
lower
coefficient
variation
compared
conventional
workflows
solvent
precipitation
or
ultrafiltration.
Journal of Proteome Research,
Год журнала:
2025,
Номер
unknown
Опубликована: Янв. 30, 2025
The
quantification
of
proteoforms,
i.e.,
all
molecular
forms
in
which
proteins
can
be
present,
by
top-down
proteomics
provides
essential
insights
into
biological
processes
at
the
level.
Isobaric
labeling-based
strategies
are
suitable
for
multidimensional
separation
and
allow
multiplexing
samples.
Here,
we
investigated
cysteine-directed
isobaric
labeling
iodoTMT
combination
with
a
gel-
gas-phase
fractionation
(GeLC-FAIMS-MS)
in-depth
quantitative
proteoform
analysis.
We
optimized
acquisition
workflow
(i.e.,
FAIMS
compensation
voltages,
isolation
windows,
strategy,
fragmentation
method)
using
two-proteome
mix
to
increase
number
quantified
proteoforms
reduce
ratio
compression.
Additionally,
implemented
mass
feature-based
strategy
widely
used
deconvolution
algorithm
FLASHDeconv,
improves
facilitates
data
GeLC-FAIMS-MS
was
applied
quantitatively
analyze
proteome
Escherichia
coli
grown
under
glucose
or
acetate
as
sole
carbon
source,
resulting
identification
726
differentially
abundant
proteoforms.
Chinese Journal of Chromatography,
Год журнала:
2024,
Номер
42(7), С. 601 - 612
Опубликована: Июль 1, 2024
Proteomics
profiling
plays
an
important
role
in
biomedical
studies.
Proteomics
studies
are
much
more
complicated
than
genome
research,
mainly
because
of
the
complexity
and
diversity
proteomic
samples.
High
performance
liquid
chromatography-mass
spectrometry
(HPLC-MS)
is
a
fundamental
tool
proteomics
research
owing
to
its
high
speed,
resolution,
sensitivity.
targets
from
peptides
individual
proteins
larger
protein
complexes,
molecular
weight
which
gradually
increases,
leading
sustained
increases
structural
compositional
alterations
properties.
Therefore,
selection
various
separation
strategies
stationary-phase
parameters
crucial
when
dealing
with
different
for
in-depth
analysis.
This
article
provides
overview
commonly
used
chromatographic-separation
laboratory,
including
reversed-phase
chromatography
(RPLC),
hydrophilic
interaction
(HILIC),
hydrophobic
(HIC),
ion-exchange
(IEC),
size-exclusion
(SEC),
as
well
their
applications
selectivity
context
biomacromolecules.
At
present,
no
single
chromatographic
or
electrophoretic
technology
features
peak
capacity
required
resolve
such
complex
mixtures
into
components.
Multidimensional
(MDLC),
combines
orthogonal
modes
MS,
research.
In
MDLC
strategy,
IEC,
together
RPLC,
remains
most
widely
mode
analysis;
other
methods
also
frequently
peptide/protein
fractionation.
technologies
variety
analyses
have
undergone
great
development.
Two
systems
profiling:
"bottom-up"
approach
"top-down"
approach.
The
"shotgun"
method
typical
strategy
that
based
on
RPLC
whole-protein-sample
digests
coupled
MS;
it
excellent
technique
identifying
large
number
proteins.
"Top-down"
analysis
intact
detailed
information;
thus,
this
may
be
advantageous
analyzing
post-translational
modifications
(PTMs)
paper,
protein-protein
(PPI)
proteome
samples
briefly
reviewed.
diverse
combinations
set
up
described,
compatibility
issues
between
mobile
phases
analytes,
multidimensional
analyzed.
Novel
developments
techniques,
high-abundance
depletion
arrays,
further
discussed.
review,
solutions
proposed
by
researchers
encountering
emphasized.
Moreover,
HPLC-MS
combined
sample
pretreatment
study
exosomal
single-cell
examined.
During
exosome
isolation,
use
ultracentrifugation
SEC
can
yield
exosomes
higher
purity.
ultra-large-pore-size
packing
materials
(200
nm)
enables
isolation
subgroups,
revealed
significant
differences
composition
function
these
subgroups.
field
proteomics,
addressed
challenges
related
reducing
processing
volumes,
preventing
loss,
avoiding
contamination
during
preparation.
Innovative
improvements,
utilization
capillaries
microchips
platforms
minimize
contact
area
droplets,
been
proposed.
integration
techniques
shows
some
progress.
summary,
focuses
recent
advances
comprehensive
reference
future
proteomics.
Clinical Proteomics,
Год журнала:
2024,
Номер
21(1)
Опубликована: Сен. 30, 2024
Cardiovascular
diseases
(CVDs)
are
the
leading
cause
of
death
worldwide,
and
prevalence
CVDs
increases
markedly
with
age.
Due
to
high
energetic
demand,
heart
is
highly
sensitive
mitochondrial
dysfunction.
The
complexity
cardiac
proteome
hinders
development
effective
strategies
that
target
dysfunction
in
CVDs.
Mammalian
mitochondria
composed
over
1000
proteins,
most
which
can
undergo
post-translational
modifications
(PTMs).
Top-down
proteomics
a
powerful
technique
for
characterizing
quantifying
proteoform
sequence
variations
PTMs.
However,
there
still
knowledge
gaps
study
age-related
changes
using
this
technique.
In
study,
we
used
top-down
identify
intact
proteoforms
young
old
hearts
determined
protein
abundance
PTMs
aging.
Research Square (Research Square),
Год журнала:
2024,
Номер
unknown
Опубликована: Янв. 18, 2024
Abstract
Cardiovascular
diseases
(CVDs)
are
the
leading
cause
of
death
worldwide,
and
prevalence
CVDs
increases
markedly
with
age.
Due
to
high
energetic
demand,
heart
is
highly
sensitive
mitochondrial
dysfunction.
The
complexity
cardiac
proteome
hinders
development
effective
strategies
that
target
dysfunction
in
CVDs.
Mammalian
mitochondria
composed
over
1000
proteins,
most
which
can
undergo
post-translational
protein
modifications
(PTMs).
Top-down
proteomics
a
powerful
technique
for
characterizing
quantifying
all
sequence
variations
PTMs.
However,
there
still
knowledge
gaps
study
age-related
proteoform
changes
using
this
technique.
In
study,
we
used
top-down
identify
intact
proteoforms
young
old
hearts
determined
abundance
PTMs
aging.
METHODS:
Intact
were
isolated
from
(4-month-old)
(24-25-month-old)
mice.
lysed,
lysates
subjected
denaturation,
reduction,
alkylation.
For
quantitative
analysis,
12
runs
total
arising
3
biological
replicates
two
conditions,
technical
duplicates
each
sample.
collected
datasets
deconvoluted
quantified,
then
identified.
RESULTS:
From
LC-MS/MS
runs,
identified
134
unique
proteins
different
sub-mitochondrial
compartments
(OMM,
IMS,
IMM,
matrix).
823
mass
ranges
Compared
mice,
7
exhibited
increased
13
decreased
Our
analysis
also
detected
proteoforms,
including
N
-terminal
acetylation,
lysine
succinylation,
oxidation,
phosphorylation.
CONCLUSION:
By
combining
enrichment
fractionation
ultrahigh-pressure
liquid
chromatography
(UPLC)-MS
label-free
quantitation,
successfully
quantified
complex
proteome.
Using
approach,
heart.
Research Square (Research Square),
Год журнала:
2024,
Номер
unknown
Опубликована: Март 20, 2024
Abstract
Top-down
proteomics
(TDP)
has
seen
significant
advances
in
the
past,
and
a
plethora
of
sample
preparation
workflows
have
been
developed.
Here,
we
systematically
investigated
influence
different
steps
on
proteoform
protein
identifications,
including
cell
lysis,
reduction
alkylation,
enrichment,
purification,
fractionation.
We
found
that
all
subset
proteoforms
identified
(e.g.,
their
number,
confidence,
physicochemical
properties,
artificially
generated
modifications).
The
various
strategies
resulted
complementary
significantly
increasing
proteome
coverage.
Overall,
13,975
from
2,720
proteins
human
Caco-2
cells
were
identified.
results
presented
can
serve
as
suggestions
for
designing
adapting
TDP
to
particular
research
questions.
Moreover,
sampling
bias
modifications
at
intact
level
will
also
be
useful
improving
bottom-up
approaches.
