41 research outputs found
Highly Sensitive Phosphoproteomics by Tailoring Solid-Phase Extraction to Electrostatic Repulsion-Hydrophilic Interaction Chromatography
In the past decade, several strategies
for comprehensive phosphoproteome
analysis have been introduced. Most of them combine different phosphopeptide
enrichment techniques and require starting material in the milligram
range, as a consequence of their insufficient sensitivity. This limitation
impairs the applicability of phosphoproteomics to a wide variety of
clinical research, where sample material is highly limited. Here we
introduce a highly sensitive and easy-to-establish 2D bottom-up strategy
for microgram-scale phosphoproteomics, based on electrostatic repulsion–hydrophilic
interaction chromatography (ERLIC), a simple solid-phase extraction
step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS
analysis. With only 100 μg of tryptic digested, nonstimulated
HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500
nonredundant and highly confident phosphorylation sites (per replicate).
We assigned all phosphorylation sites to 3013 phosphoproteins, covering
the entire dynamic range from 10<sup>7</sup> down to a few copies
per cell. Compared to affinity-based-enrichment methods using Ti<sup>4+</sup>, our ERLIC-based strategy enriched considerably longer and
more acidic phosphopeptides and consequently, we identified 327 phosphorylated
C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS
render its future promising for microgram-scale-phosphoproteomics
in biological, biomedical, and clinical research
Highly Sensitive Phosphoproteomics by Tailoring Solid-Phase Extraction to Electrostatic Repulsion-Hydrophilic Interaction Chromatography
In the past decade, several strategies
for comprehensive phosphoproteome
analysis have been introduced. Most of them combine different phosphopeptide
enrichment techniques and require starting material in the milligram
range, as a consequence of their insufficient sensitivity. This limitation
impairs the applicability of phosphoproteomics to a wide variety of
clinical research, where sample material is highly limited. Here we
introduce a highly sensitive and easy-to-establish 2D bottom-up strategy
for microgram-scale phosphoproteomics, based on electrostatic repulsion–hydrophilic
interaction chromatography (ERLIC), a simple solid-phase extraction
step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS
analysis. With only 100 μg of tryptic digested, nonstimulated
HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500
nonredundant and highly confident phosphorylation sites (per replicate).
We assigned all phosphorylation sites to 3013 phosphoproteins, covering
the entire dynamic range from 10<sup>7</sup> down to a few copies
per cell. Compared to affinity-based-enrichment methods using Ti<sup>4+</sup>, our ERLIC-based strategy enriched considerably longer and
more acidic phosphopeptides and consequently, we identified 327 phosphorylated
C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS
render its future promising for microgram-scale-phosphoproteomics
in biological, biomedical, and clinical research
iTRAQ Analysis of a Cell Culture Model for Malignant Transformation, Including Comparison with 2D-PAGE and SILAC
To study human cancer development, cell culture models
for malignant
transformation can be used. In 1999 Hahn and Coworkers introduced
such a model system and established herewith a basis for research
on human tumorigenesis. Primary human fibroblasts are sequentially
transduced with defined genetic elements (hTERT, SV40 ER, and H-Ras<sup>V12</sup>), resulting in four defined cell lines, whereby the last
has a fully transformed phenotype. In order to get a deeper insight
into the molecular biology of human tumorigenesis, we compared the
proteomes of these four cell lines following a multimethod concept.
At the beginning we assumed SILAC and sample fractionation with COFRADIC
is the method of choice to analyze the cell culture model for malignant
transformation. Here, the compared samples are combined before sample
preparation, thus avoiding differences in sample preparation, and
using COFRADIC notably reduces sample complexity. Because 2D-PAGE
is a standard method for the separation and visualization of closely
related proteomes, we decided to analyze and compare the proteomes
of these four cell lines in a first approach by differential 2D-PAGE.
Surprisingly, we discovered much more unique results with iTRAQ and
sample fractionation with SCX than with the combination of 2D-PAGE
and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies
not only in number of yielded results but also in analysis time. Here,
we present the iTRAQ quantification results and compare them with
the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the
protein level at each transition. Thereby, SV40 has the strongest
impact on the proteome. In detail we identified 201 regulated proteins.
Beside others, these proteins are involved in cytoskeleton, RNA processing,
and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins.
For example, MCM proteins are up-regulated and collagens are down-regulated
due to SV40 ER expression. Furthermore we made the observation that
proteins containing the same domain have analogous regulation profiles
during malignant transformation. For instance, several proteins containing
a CH or LIM domain are down-regulated. Moreover, by this study and
the defined cell culture model, changes could be clearly matched to
specific steps during tumorigenesis
Enrichment of Cross-Linked Peptides Using Charge-Based Fractional Diagonal Chromatography (ChaFRADIC)
Chemical cross-linking of proteins
is an emerging field with huge
potential for the structural investigation of proteins and protein
complexes. Owing to the often relatively low yield of cross-linking
products, their identification in complex samples benefits from enrichment
procedures prior to mass spectrometry analysis. So far, this is mainly
accomplished by using biotin moieties in specific cross-linkers or
by applying strong cation exchange chromatography (SCX) for a relatively
crude enrichment. We present a novel workflow to enrich cross-linked
peptides by utilizing charge-based fractional diagonal chromatography
(ChaFRADIC). On the basis of two-dimensional diagonal SCX separation,
we could increase the number of identified cross-linked peptides for
samples of different complexity: pure cross-linked BSA, cross-linked
BSA spiked into a simple protein mixture, and cross-linked BSA spiked
into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion
chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC
approach is straightforward, reproducible, and independent of the
cross-linking chemistry and cross-linker properties
iTRAQ Analysis of a Cell Culture Model for Malignant Transformation, Including Comparison with 2D-PAGE and SILAC
To study human cancer development, cell culture models
for malignant
transformation can be used. In 1999 Hahn and Coworkers introduced
such a model system and established herewith a basis for research
on human tumorigenesis. Primary human fibroblasts are sequentially
transduced with defined genetic elements (hTERT, SV40 ER, and H-Ras<sup>V12</sup>), resulting in four defined cell lines, whereby the last
has a fully transformed phenotype. In order to get a deeper insight
into the molecular biology of human tumorigenesis, we compared the
proteomes of these four cell lines following a multimethod concept.
At the beginning we assumed SILAC and sample fractionation with COFRADIC
is the method of choice to analyze the cell culture model for malignant
transformation. Here, the compared samples are combined before sample
preparation, thus avoiding differences in sample preparation, and
using COFRADIC notably reduces sample complexity. Because 2D-PAGE
is a standard method for the separation and visualization of closely
related proteomes, we decided to analyze and compare the proteomes
of these four cell lines in a first approach by differential 2D-PAGE.
Surprisingly, we discovered much more unique results with iTRAQ and
sample fractionation with SCX than with the combination of 2D-PAGE
and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies
not only in number of yielded results but also in analysis time. Here,
we present the iTRAQ quantification results and compare them with
the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the
protein level at each transition. Thereby, SV40 has the strongest
impact on the proteome. In detail we identified 201 regulated proteins.
Beside others, these proteins are involved in cytoskeleton, RNA processing,
and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins.
For example, MCM proteins are up-regulated and collagens are down-regulated
due to SV40 ER expression. Furthermore we made the observation that
proteins containing the same domain have analogous regulation profiles
during malignant transformation. For instance, several proteins containing
a CH or LIM domain are down-regulated. Moreover, by this study and
the defined cell culture model, changes could be clearly matched to
specific steps during tumorigenesis
iTRAQ Analysis of a Cell Culture Model for Malignant Transformation, Including Comparison with 2D-PAGE and SILAC
To study human cancer development, cell culture models
for malignant
transformation can be used. In 1999 Hahn and Coworkers introduced
such a model system and established herewith a basis for research
on human tumorigenesis. Primary human fibroblasts are sequentially
transduced with defined genetic elements (hTERT, SV40 ER, and H-Ras<sup>V12</sup>), resulting in four defined cell lines, whereby the last
has a fully transformed phenotype. In order to get a deeper insight
into the molecular biology of human tumorigenesis, we compared the
proteomes of these four cell lines following a multimethod concept.
At the beginning we assumed SILAC and sample fractionation with COFRADIC
is the method of choice to analyze the cell culture model for malignant
transformation. Here, the compared samples are combined before sample
preparation, thus avoiding differences in sample preparation, and
using COFRADIC notably reduces sample complexity. Because 2D-PAGE
is a standard method for the separation and visualization of closely
related proteomes, we decided to analyze and compare the proteomes
of these four cell lines in a first approach by differential 2D-PAGE.
Surprisingly, we discovered much more unique results with iTRAQ and
sample fractionation with SCX than with the combination of 2D-PAGE
and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies
not only in number of yielded results but also in analysis time. Here,
we present the iTRAQ quantification results and compare them with
the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the
protein level at each transition. Thereby, SV40 has the strongest
impact on the proteome. In detail we identified 201 regulated proteins.
Beside others, these proteins are involved in cytoskeleton, RNA processing,
and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins.
For example, MCM proteins are up-regulated and collagens are down-regulated
due to SV40 ER expression. Furthermore we made the observation that
proteins containing the same domain have analogous regulation profiles
during malignant transformation. For instance, several proteins containing
a CH or LIM domain are down-regulated. Moreover, by this study and
the defined cell culture model, changes could be clearly matched to
specific steps during tumorigenesis
iTRAQ Analysis of a Cell Culture Model for Malignant Transformation, Including Comparison with 2D-PAGE and SILAC
To study human cancer development, cell culture models
for malignant
transformation can be used. In 1999 Hahn and Coworkers introduced
such a model system and established herewith a basis for research
on human tumorigenesis. Primary human fibroblasts are sequentially
transduced with defined genetic elements (hTERT, SV40 ER, and H-Ras<sup>V12</sup>), resulting in four defined cell lines, whereby the last
has a fully transformed phenotype. In order to get a deeper insight
into the molecular biology of human tumorigenesis, we compared the
proteomes of these four cell lines following a multimethod concept.
At the beginning we assumed SILAC and sample fractionation with COFRADIC
is the method of choice to analyze the cell culture model for malignant
transformation. Here, the compared samples are combined before sample
preparation, thus avoiding differences in sample preparation, and
using COFRADIC notably reduces sample complexity. Because 2D-PAGE
is a standard method for the separation and visualization of closely
related proteomes, we decided to analyze and compare the proteomes
of these four cell lines in a first approach by differential 2D-PAGE.
Surprisingly, we discovered much more unique results with iTRAQ and
sample fractionation with SCX than with the combination of 2D-PAGE
and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies
not only in number of yielded results but also in analysis time. Here,
we present the iTRAQ quantification results and compare them with
the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the
protein level at each transition. Thereby, SV40 has the strongest
impact on the proteome. In detail we identified 201 regulated proteins.
Beside others, these proteins are involved in cytoskeleton, RNA processing,
and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins.
For example, MCM proteins are up-regulated and collagens are down-regulated
due to SV40 ER expression. Furthermore we made the observation that
proteins containing the same domain have analogous regulation profiles
during malignant transformation. For instance, several proteins containing
a CH or LIM domain are down-regulated. Moreover, by this study and
the defined cell culture model, changes could be clearly matched to
specific steps during tumorigenesis
iTRAQ Analysis of a Cell Culture Model for Malignant Transformation, Including Comparison with 2D-PAGE and SILAC
To study human cancer development, cell culture models
for malignant
transformation can be used. In 1999 Hahn and Coworkers introduced
such a model system and established herewith a basis for research
on human tumorigenesis. Primary human fibroblasts are sequentially
transduced with defined genetic elements (hTERT, SV40 ER, and H-Ras<sup>V12</sup>), resulting in four defined cell lines, whereby the last
has a fully transformed phenotype. In order to get a deeper insight
into the molecular biology of human tumorigenesis, we compared the
proteomes of these four cell lines following a multimethod concept.
At the beginning we assumed SILAC and sample fractionation with COFRADIC
is the method of choice to analyze the cell culture model for malignant
transformation. Here, the compared samples are combined before sample
preparation, thus avoiding differences in sample preparation, and
using COFRADIC notably reduces sample complexity. Because 2D-PAGE
is a standard method for the separation and visualization of closely
related proteomes, we decided to analyze and compare the proteomes
of these four cell lines in a first approach by differential 2D-PAGE.
Surprisingly, we discovered much more unique results with iTRAQ and
sample fractionation with SCX than with the combination of 2D-PAGE
and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies
not only in number of yielded results but also in analysis time. Here,
we present the iTRAQ quantification results and compare them with
the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the
protein level at each transition. Thereby, SV40 has the strongest
impact on the proteome. In detail we identified 201 regulated proteins.
Beside others, these proteins are involved in cytoskeleton, RNA processing,
and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins.
For example, MCM proteins are up-regulated and collagens are down-regulated
due to SV40 ER expression. Furthermore we made the observation that
proteins containing the same domain have analogous regulation profiles
during malignant transformation. For instance, several proteins containing
a CH or LIM domain are down-regulated. Moreover, by this study and
the defined cell culture model, changes could be clearly matched to
specific steps during tumorigenesis
iTRAQ Analysis of a Cell Culture Model for Malignant Transformation, Including Comparison with 2D-PAGE and SILAC
To study human cancer development, cell culture models
for malignant
transformation can be used. In 1999 Hahn and Coworkers introduced
such a model system and established herewith a basis for research
on human tumorigenesis. Primary human fibroblasts are sequentially
transduced with defined genetic elements (hTERT, SV40 ER, and H-Ras<sup>V12</sup>), resulting in four defined cell lines, whereby the last
has a fully transformed phenotype. In order to get a deeper insight
into the molecular biology of human tumorigenesis, we compared the
proteomes of these four cell lines following a multimethod concept.
At the beginning we assumed SILAC and sample fractionation with COFRADIC
is the method of choice to analyze the cell culture model for malignant
transformation. Here, the compared samples are combined before sample
preparation, thus avoiding differences in sample preparation, and
using COFRADIC notably reduces sample complexity. Because 2D-PAGE
is a standard method for the separation and visualization of closely
related proteomes, we decided to analyze and compare the proteomes
of these four cell lines in a first approach by differential 2D-PAGE.
Surprisingly, we discovered much more unique results with iTRAQ and
sample fractionation with SCX than with the combination of 2D-PAGE
and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies
not only in number of yielded results but also in analysis time. Here,
we present the iTRAQ quantification results and compare them with
the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the
protein level at each transition. Thereby, SV40 has the strongest
impact on the proteome. In detail we identified 201 regulated proteins.
Beside others, these proteins are involved in cytoskeleton, RNA processing,
and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins.
For example, MCM proteins are up-regulated and collagens are down-regulated
due to SV40 ER expression. Furthermore we made the observation that
proteins containing the same domain have analogous regulation profiles
during malignant transformation. For instance, several proteins containing
a CH or LIM domain are down-regulated. Moreover, by this study and
the defined cell culture model, changes could be clearly matched to
specific steps during tumorigenesis
iTRAQ Analysis of a Cell Culture Model for Malignant Transformation, Including Comparison with 2D-PAGE and SILAC
To study human cancer development, cell culture models
for malignant
transformation can be used. In 1999 Hahn and Coworkers introduced
such a model system and established herewith a basis for research
on human tumorigenesis. Primary human fibroblasts are sequentially
transduced with defined genetic elements (hTERT, SV40 ER, and H-Ras<sup>V12</sup>), resulting in four defined cell lines, whereby the last
has a fully transformed phenotype. In order to get a deeper insight
into the molecular biology of human tumorigenesis, we compared the
proteomes of these four cell lines following a multimethod concept.
At the beginning we assumed SILAC and sample fractionation with COFRADIC
is the method of choice to analyze the cell culture model for malignant
transformation. Here, the compared samples are combined before sample
preparation, thus avoiding differences in sample preparation, and
using COFRADIC notably reduces sample complexity. Because 2D-PAGE
is a standard method for the separation and visualization of closely
related proteomes, we decided to analyze and compare the proteomes
of these four cell lines in a first approach by differential 2D-PAGE.
Surprisingly, we discovered much more unique results with iTRAQ and
sample fractionation with SCX than with the combination of 2D-PAGE
and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies
not only in number of yielded results but also in analysis time. Here,
we present the iTRAQ quantification results and compare them with
the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the
protein level at each transition. Thereby, SV40 has the strongest
impact on the proteome. In detail we identified 201 regulated proteins.
Beside others, these proteins are involved in cytoskeleton, RNA processing,
and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins.
For example, MCM proteins are up-regulated and collagens are down-regulated
due to SV40 ER expression. Furthermore we made the observation that
proteins containing the same domain have analogous regulation profiles
during malignant transformation. For instance, several proteins containing
a CH or LIM domain are down-regulated. Moreover, by this study and
the defined cell culture model, changes could be clearly matched to
specific steps during tumorigenesis