13 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
Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos
A variety of quantitative proteomics methods have been
developed,
including label-free, metabolic labeling, and isobaric chemical labeling
using iTRAQ or TMT. Here, these methods were compared in terms of
the depth of proteome coverage, quantification accuracy, precision,
and reproducibility using a high-performance hybrid mass spectrometer,
LTQ Orbitrap Velos. Our results show that (1) the spectral counting
method provides the deepest proteome coverage for identification,
but its quantification performance is worse than labeling-based approaches,
especially the quantification reproducibility; (2) metabolic labeling
and isobaric chemical labeling are capable of accurate, precise, and
reproducible quantification and provide deep proteome coverage for
quantification; isobaric chemical labeling surpasses metabolic labeling
in terms of quantification precision and reproducibility; and (3)
iTRAQ and TMT perform similarly in all aspects compared in the current
study using a CID-HCD dual scan configuration. On the basis of the
unique advantages of each method, we provide guidance for selection
of the appropriate method for a quantitative proteomics study
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
Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos
A variety of quantitative proteomics methods have been
developed,
including label-free, metabolic labeling, and isobaric chemical labeling
using iTRAQ or TMT. Here, these methods were compared in terms of
the depth of proteome coverage, quantification accuracy, precision,
and reproducibility using a high-performance hybrid mass spectrometer,
LTQ Orbitrap Velos. Our results show that (1) the spectral counting
method provides the deepest proteome coverage for identification,
but its quantification performance is worse than labeling-based approaches,
especially the quantification reproducibility; (2) metabolic labeling
and isobaric chemical labeling are capable of accurate, precise, and
reproducible quantification and provide deep proteome coverage for
quantification; isobaric chemical labeling surpasses metabolic labeling
in terms of quantification precision and reproducibility; and (3)
iTRAQ and TMT perform similarly in all aspects compared in the current
study using a CID-HCD dual scan configuration. On the basis of the
unique advantages of each method, we provide guidance for selection
of the appropriate method for a quantitative proteomics study
Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos
A variety of quantitative proteomics methods have been
developed,
including label-free, metabolic labeling, and isobaric chemical labeling
using iTRAQ or TMT. Here, these methods were compared in terms of
the depth of proteome coverage, quantification accuracy, precision,
and reproducibility using a high-performance hybrid mass spectrometer,
LTQ Orbitrap Velos. Our results show that (1) the spectral counting
method provides the deepest proteome coverage for identification,
but its quantification performance is worse than labeling-based approaches,
especially the quantification reproducibility; (2) metabolic labeling
and isobaric chemical labeling are capable of accurate, precise, and
reproducible quantification and provide deep proteome coverage for
quantification; isobaric chemical labeling surpasses metabolic labeling
in terms of quantification precision and reproducibility; and (3)
iTRAQ and TMT perform similarly in all aspects compared in the current
study using a CID-HCD dual scan configuration. On the basis of the
unique advantages of each method, we provide guidance for selection
of the appropriate method for a quantitative proteomics study
Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in <i>Arabidopsis</i>
Strigolactones
(SLs) are a new class of plant hormones. In addition
to acting as a key inhibitor of shoot branching, SLs stimulate seed
germination of root parasitic plants and promote hyphal branching
and root colonization of symbiotic arbuscular mycorrhizal fungi. They
also regulate many other aspects of plant growth and development.
At the transcription level, SL-regulated genes have been reported.
However, nothing is known about the proteome regulated by this new
class of plant hormones. A quantitative proteomics approach using
an isobaric chemical labeling reagent, iTRAQ, to identify the proteome
regulated by SLs in <i>Arabidopsis</i> seedlings is presented.
It was found that SLs regulate the expression of about three dozen
proteins that have not been previously assigned to SL pathways. These
findings provide a new tool to investigate the molecular mechanism
of action of SLs