Most
drug
candidates
in
the
development
pipeline
fail
during
Phase
II
clinical
trials
because
of
inefficacy
or
unacceptable
safety
profiles.
The
costly
financial
burden
failed
late-stage
may
be
transferred
to
few
drugs
that
successfully
navigate
approval
process.
efficient
discovery
and
therapeutic
require
deep
profiling
interactions
between
their
biological
targets
for
gaining
a
critical
understanding
mechanisms
while
limiting
unwanted
binding
other
(off-target)
proteins,
which
can
lead
adverse
reactions
toxic
effects.
Thermal
proteome
(TPP)
mass
spectrometry-based
cellular
thermal
shift
assay
(CETSA
MS)
are
adaptations
CETSA
technique,
exploit
phenomenon
ligand-induced
stabilization
proteins.
Collectively
referred
as
stability
assays
(MS-TSA),
they
have
refined
process
target
profiling.
While
rapidly
evolving
MS-TSA
techniques
provided
remarkable
insights
into
drug-protein
interactome,
challenges
remain,
particularly
obtaining
melting
curves
from
low-abundance
poorly
soluble
objective
this
dissertation
is
innovate
conventional
approaches
with
novel
combinations
MS-enabling
technologies
enhanced
performance.
We
specifically
focused
on
improving
characterization
profiles
proteins
low
abundance
soluble.
Chapter
1
presents
historical
overview
tools
preceded
current
drug-target
techniques,
provides
context
insight
about
modern
setting.
A
thorough
review
discussing
evolution
impact
pharmaceutical
research
also
included
1.
Chapters
2
3
assess
integration
tequnique.
In
2,
we
introduced
improved
MS-based
acquisition
(iMAATSA),
combine
several
technologies.
combination
demonstrated
synergistic
effect
performance
by
increased
protein
identifications
high-quality
using
model
system.
Aspects
iMAATSA
were
further
developed
3,
where
membrane-enriched
stable
isotope
isobaric-labeled
carrier
channel
(meSIILCC)
approach
was
detection
membrane
MS-TSA.
After
testing
four
protein-enrichment
protocols,
commercially
available
kit
selected
improve
meSIILCC
workflow.
separate
DMSO-only
control
experiment
optimized
conducted.
significant
increase
number
identified
unique
quantified
peptides
per
annotated
"plasma
membrane"
approach.
Also,
acquired
results
unsuitable
isobaric
tag
channels
due
interference.
final
Chapter,
focus
how
could
utilized
future
highlight
aspects
need
developed.
--Author's
abstract
ACS Measurement Science Au,
Год журнала:
2024,
Номер
4(4), С. 338 - 417
Опубликована: Июнь 4, 2024
Proteomics
is
the
large
scale
study
of
protein
structure
and
function
from
biological
systems
through
identification
quantification."Shotgun
proteomics"
or
"bottom-up
prevailing
strategy,
in
which
proteins
are
hydrolyzed
into
peptides
that
analyzed
by
mass
spectrometry.Proteomics
studies
can
be
applied
to
diverse
ranging
simple
proteoforms,
protein-protein
interactions,
structural
alterations,
absolute
relative
quantification,
post-translational
modifications,
stability.To
enable
this
range
different
experiments,
there
strategies
for
proteome
analysis.The
nuances
how
proteomic
workflows
differ
may
challenging
understand
new
practitioners.Here,
we
provide
a
comprehensive
overview
proteomics
methods.We
cover
biochemistry
basics
extraction
interpretation
orthogonal
validation.We
expect
Review
will
serve
as
handbook
researchers
who
field
bottom-up
proteomics.
Nature Communications,
Год журнала:
2024,
Номер
15(1)
Опубликована: Фев. 3, 2024
Comprehensive
proteomic
analysis
is
essential
to
elucidate
molecular
pathways
and
protein
functions.
Despite
tremendous
progress
in
proteomics,
current
studies
still
suffer
from
limited
coverage
dynamic
range.
Here,
we
utilize
micropillar
array
columns
(µPACs)
together
with
wide-window
acquisition
the
AI-based
CHIMERYS
search
engine
achieve
excellent
comprehensiveness
for
bulk
affinity
purification
mass
spectrometry
single
cell
proteomics.
Our
data
show
that
µPACs
identify
≤50%
more
peptides
≤24%
proteins,
while
offering
improved
throughput,
which
critical
large
(clinical)
proteomics
studies.
Combining
wide
precursor
isolation
widths
of
m/z
4-12
identified
+51-74%
+59-150%
proteins
peptides,
respectively,
cell,
co-immunoprecipitation,
multi-species
samples
over
a
conventional
workflow
at
well-controlled
false
discovery
rates.
The
further
offers
precision,
CVs
<7%
low
input
samples,
accuracy,
deviations
<10%
expected
fold
changes
regular
abundance
two-proteome
mixes.
Compared
workflow,
our
entire
optimized
platform
discovered
92%
potential
interactors
protein-protein
interaction
study
on
chromatin
remodeler
Smarca5/Snf2h.
These
include
previously
described
Smarca5
binding
partners
undescribed
ones
including
Arid1a,
another
key
roles
neurodevelopmental
malignant
disorders.
Journal of the American Society for Mass Spectrometry,
Год журнала:
2023,
Номер
34(10), С. 2374 - 2380
Опубликована: Авг. 18, 2023
Single-cell
proteomics
(SCP)
can
provide
information
that
is
unattainable
through
either
bulk-scale
protein
measurements
or
single-cell
profiling
of
other
omes.
Maximizing
proteome
coverage
often
requires
custom
instrumentation,
consumables,
and
reagents
for
sample
processing
separations,
which
has
limited
the
accessibility
SCP
to
a
small
number
specialized
laboratories.
Commercial
platforms
have
become
available
cell
isolation
preparation,
but
high
cost
these
technical
expertise
required
their
operation
place
them
out
reach
many
interested
Here,
we
assessed
new
HP
D100
Single
Cell
Dispenser
label-free
SCP.
The
low-cost
instrument
proved
highly
accurate
reproducible
dispensing
in
range
from
200
nL
2
μL.
We
used
isolate
prepare
single
cells
within
384-well
PCR
plates.
When
well
plates
were
immediately
centrifuged
following
again
after
reagent
dispensing,
found
∼97%
wells
identified
software
as
containing
indeed
provided
expected
cell.
This
commercial
dispenser
combined
with
one-step
provides
very
rapid
easy-to-use
workflow
no
reduction
relative
nanowell-based
workflow,
also
facilitate
autosampling
unmodified
instrumentation.
samples
analyzed
using
home-packed
30
μm
i.d.
nanoLC
columns
commercially
50
columns.
resulted
∼35%
fewer
proteins.
However,
plate-based
preparation
platform,
presented
fully
relatively
alternative
separation,
should
greatly
broaden
Journal of Proteome Research,
Год журнала:
2024,
Номер
23(8), С. 2934 - 2947
Опубликована: Янв. 22, 2024
Intelligent
data
acquisition
(IDA)
strategies,
such
as
a
real-time
database
search
(RTS),
have
improved
the
depth
of
proteome
coverage
for
experiments
that
utilize
isobaric
labels
and
gas
phase
purification
techniques
(i.e.,
SPS-MS3).
In
this
work,
we
introduce
inSeqAPI,
an
instrument
application
programing
interface
(iAPI)
program
enables
construction
novel
algorithms.
First,
analyze
biotinylated
cysteine
peptides
from
ABPP
to
demonstrate
method
within
inSeqAPI
performs
similarly
equivalent
vendor
method.
Then,
describe
PairQuant,
designed
hyperplexing
approach
utilizes
protein-level
isotopic
labeling
peptide-level
TMT
labeling.
PairQuant
allows
analysis
36
conditions
in
single
sample
achieves
∼98%
both
peptide
pair
partners
hyperplexed
experiment
well
40%
improvement
number
quantified
sites
compared
with
non-RTS
acquisition.
We
applied
study
ligandable
nucleus
leading
identification
additional
druggable
on
protein-
DNA-interaction
domains
transcription
regulators
nuclear
ubiquitin
ligases.
ACS Measurement Science Au,
Год журнала:
2024,
Номер
4(4), С. 315 - 337
Опубликована: Апрель 15, 2024
Recent
advancements
in
mass
spectrometry
(MS)
have
revolutionized
quantitative
proteomics,
with
multiplex
isotope
labeling
emerging
as
a
key
strategy
for
enhancing
accuracy,
precision,
and
throughput.
This
tutorial
review
offers
comprehensive
overview
of
techniques,
including
precursor-based,
defect-based,
reporter
ion-based,
hybrid
methods.
It
details
their
fundamental
principles,
advantages,
inherent
limitations
along
strategies
to
mitigate
the
limitation
ratio-distortion.
will
also
cover
applications
latest
progress
these
techniques
across
various
domains,
cancer
biomarker
discovery,
neuroproteomics,
post-translational
modification
analysis,
cross-linking
MS,
single-cell
proteomics.
Review
aims
provide
guidance
researchers
on
selecting
appropriate
methods
specific
goals
while
highlighting
potential
future
directions
this
rapidly
evolving
field.
Analytical Chemistry,
Год журнала:
2025,
Номер
unknown
Опубликована: Янв. 6, 2025
Single-cell
proteomics
(SCP)
detected
based
on
different
technologies
always
involves
batch-specific
variations
because
of
differences
in
sample
processing
and
other
potential
biases.
How
to
integrate
SCP
data
effectively
has
become
a
great
challenge.
Integration
not
only
requires
the
conservation
true
biological
variances,
but
also
realizes
removal
unwanted
batch
effects.
In
this
study,
benchmarking
analysis
popular
integration
methods
was
conducted
determine
most
suitable
method
for
data.
To
comprehensively
evaluate
performance
these
methods,
novel
evaluation
system
proposed
integrating
This
consists
three
objective
measures
from
perspectives:
category
(a),
efficacy
correcting
effects;
(b),
power
conserving
variances;
(c),
ability
identify
consistent
markers.
For
comprehensive
evaluation,
five
benchmark
sets
under
scenarios
(containing
substantial
proteins,
cells,
multiple
batches,
cell
types,
unbalanced
data)
were
utilized
selecting
method.
As
result,
ComBat,
Scanorama,
Seurat
version
3
CCA,
identified
as
recommended
Overall,
systematic
might
provide
valuable
guidance
choosing
appropriate
SCP.
Journal of Proteome Research,
Год журнала:
2024,
Номер
unknown
Опубликована: Май 7, 2024
Single-cell
proteomics
is
a
powerful
approach
to
precisely
profile
protein
landscapes
within
individual
cells
toward
comprehensive
understanding
of
proteomic
functions
and
tissue
cellular
states.
The
inherent
challenges
associated
with
limited
starting
material
demand
heightened
analytical
sensitivity.
Just
as
advances
in
sample
preparation
maximize
the
amount
that
makes
it
from
cell
mass
spectrometer,
we
strive
number
ions
make
ion
source
detector.
In
isobaric
tagging
experiments,
reporter
generation
limits
quantitative
accuracy
precision.
combination
infrared
photoactivation
parking
circumvents
