7 research outputs found
Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization
The
expanding use of surfactants for proteome sample preparations
has prompted the need to systematically optimize the application and
removal of these MS-deleterious agents prior to proteome measurements.
Here we compare four detergent cleanup methods (trichloroacetic acid
(TCA) precipitation, chloroform/methanol/water (CMW) extraction, a
commercial detergent removal spin column method (DRS) and filter-aided
sample preparation (FASP)) to provide efficiency benchmarks with respect
to protein, peptide, and spectral identifications in each case. Our
results show that for protein-limited samples, FASP outperforms the
other three cleanup methods, while at high protein amounts, all the
methods are comparable. This information was used to investigate and
contrast molecular weight-based fractionated with unfractionated lysates
from three increasingly complex samples (Escherichia
coli K-12, a five microbial isolate mixture, and a
natural microbial community groundwater sample), all of which were
prepared with an SDS-FASP approach. The additional fractionation step
enhanced the number of protein identifications by 8% to 25% over the
unfractionated approach across the three samples
Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization
The
expanding use of surfactants for proteome sample preparations
has prompted the need to systematically optimize the application and
removal of these MS-deleterious agents prior to proteome measurements.
Here we compare four detergent cleanup methods (trichloroacetic acid
(TCA) precipitation, chloroform/methanol/water (CMW) extraction, a
commercial detergent removal spin column method (DRS) and filter-aided
sample preparation (FASP)) to provide efficiency benchmarks with respect
to protein, peptide, and spectral identifications in each case. Our
results show that for protein-limited samples, FASP outperforms the
other three cleanup methods, while at high protein amounts, all the
methods are comparable. This information was used to investigate and
contrast molecular weight-based fractionated with unfractionated lysates
from three increasingly complex samples (Escherichia
coli K-12, a five microbial isolate mixture, and a
natural microbial community groundwater sample), all of which were
prepared with an SDS-FASP approach. The additional fractionation step
enhanced the number of protein identifications by 8% to 25% over the
unfractionated approach across the three samples
Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization
The
expanding use of surfactants for proteome sample preparations
has prompted the need to systematically optimize the application and
removal of these MS-deleterious agents prior to proteome measurements.
Here we compare four detergent cleanup methods (trichloroacetic acid
(TCA) precipitation, chloroform/methanol/water (CMW) extraction, a
commercial detergent removal spin column method (DRS) and filter-aided
sample preparation (FASP)) to provide efficiency benchmarks with respect
to protein, peptide, and spectral identifications in each case. Our
results show that for protein-limited samples, FASP outperforms the
other three cleanup methods, while at high protein amounts, all the
methods are comparable. This information was used to investigate and
contrast molecular weight-based fractionated with unfractionated lysates
from three increasingly complex samples (Escherichia
coli K-12, a five microbial isolate mixture, and a
natural microbial community groundwater sample), all of which were
prepared with an SDS-FASP approach. The additional fractionation step
enhanced the number of protein identifications by 8% to 25% over the
unfractionated approach across the three samples
Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization
The
expanding use of surfactants for proteome sample preparations
has prompted the need to systematically optimize the application and
removal of these MS-deleterious agents prior to proteome measurements.
Here we compare four detergent cleanup methods (trichloroacetic acid
(TCA) precipitation, chloroform/methanol/water (CMW) extraction, a
commercial detergent removal spin column method (DRS) and filter-aided
sample preparation (FASP)) to provide efficiency benchmarks with respect
to protein, peptide, and spectral identifications in each case. Our
results show that for protein-limited samples, FASP outperforms the
other three cleanup methods, while at high protein amounts, all the
methods are comparable. This information was used to investigate and
contrast molecular weight-based fractionated with unfractionated lysates
from three increasingly complex samples (Escherichia
coli K-12, a five microbial isolate mixture, and a
natural microbial community groundwater sample), all of which were
prepared with an SDS-FASP approach. The additional fractionation step
enhanced the number of protein identifications by 8% to 25% over the
unfractionated approach across the three samples
Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization
The
expanding use of surfactants for proteome sample preparations
has prompted the need to systematically optimize the application and
removal of these MS-deleterious agents prior to proteome measurements.
Here we compare four detergent cleanup methods (trichloroacetic acid
(TCA) precipitation, chloroform/methanol/water (CMW) extraction, a
commercial detergent removal spin column method (DRS) and filter-aided
sample preparation (FASP)) to provide efficiency benchmarks with respect
to protein, peptide, and spectral identifications in each case. Our
results show that for protein-limited samples, FASP outperforms the
other three cleanup methods, while at high protein amounts, all the
methods are comparable. This information was used to investigate and
contrast molecular weight-based fractionated with unfractionated lysates
from three increasingly complex samples (Escherichia
coli K-12, a five microbial isolate mixture, and a
natural microbial community groundwater sample), all of which were
prepared with an SDS-FASP approach. The additional fractionation step
enhanced the number of protein identifications by 8% to 25% over the
unfractionated approach across the three samples
Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization
The
expanding use of surfactants for proteome sample preparations
has prompted the need to systematically optimize the application and
removal of these MS-deleterious agents prior to proteome measurements.
Here we compare four detergent cleanup methods (trichloroacetic acid
(TCA) precipitation, chloroform/methanol/water (CMW) extraction, a
commercial detergent removal spin column method (DRS) and filter-aided
sample preparation (FASP)) to provide efficiency benchmarks with respect
to protein, peptide, and spectral identifications in each case. Our
results show that for protein-limited samples, FASP outperforms the
other three cleanup methods, while at high protein amounts, all the
methods are comparable. This information was used to investigate and
contrast molecular weight-based fractionated with unfractionated lysates
from three increasingly complex samples (Escherichia
coli K-12, a five microbial isolate mixture, and a
natural microbial community groundwater sample), all of which were
prepared with an SDS-FASP approach. The additional fractionation step
enhanced the number of protein identifications by 8% to 25% over the
unfractionated approach across the three samples
Activity-Based Protein Profiling Shows Heterogeneous Signaling Adaptations to BRAF Inhibition
Patients with BRAF V600E mutant melanoma
are typically treated
with targeted BRAF kinase inhibitors, such as vemurafenib and dabrafenib.
Although these drugs are initially effective, they are not curative.
Most of the focus to date has been upon genetic mechanisms of acquired
resistance; therefore, we must better understand the global signaling
adaptations that mediate escape from BRAF inhibition. In the current
study, we have used activity-based protein profiling (ABPP) with ATP-analogue
probes to enrich kinases and other enzyme classes that contribute
to BRAF inhibitor (BRAFi) resistance in four paired isogenic BRAFi-naïve/resistant
cell line models. Our analysis showed these cell line models, which
also differ in their PTEN status, have considerable heterogeneity
in their kinase ATP probe uptake in comparing both naïve cells
and adaptations to chronic drug exposure. A number of kinases including
FAK1, SLK, and TAOK2 had increased ATP probe uptake in BRAFi resistant
cells, while KHS1 (M4K5) and BRAF had decreased ATP probe uptake in
the BRAFi-resistant cells. Gene ontology (GO) enrichment analysis
revealed BRAFi resistance is associated with a significant enhancement
in ATP probe uptake in proteins implicated in cytoskeletal organization
and adhesion, and decreases in ATP probe uptake in proteins associated
with cell metabolic processes. The ABPP approach was able to identify
key phenotypic mediators critical for each BRAFi resistant cell line.
Together, these data show that common phenotypic adaptations to BRAF
inhibition can be mediated through very different signaling networks,
suggesting considerable redundancy within the signaling of <i>BRAF</i> mutant melanoma cells