12 research outputs found
Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity
Kinases
are involved in the regulation of many cellular processes
and aberrant kinase signaling has been implicated in human disease.
As a consequence, kinases are attractive drug targets. Assessing kinase
function and drug selectivity in a more physiological context is challenging
and often hampered by the generally low expression level of kinases
and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained
attention because they allow the profiling of hundreds of kinases
against one drug at the same time. Here, we directly compared two
such methods, notably, immobilized broad spectrum kinase inhibitors
(kinobeads) and active site labeling using desthiobiotin-ATP and -ADP
probes. Affinity purification of ∼100 kinases by either kinobeads
or ATP/ADP probes was readily achieved using 1 mg of cellular protein.
Bioinformatic analysis revealed a high degree of complementarity of
the two techniques. Kinobeads covered the Tyrosine Kinase family particularly
well and ATP probes enriched higher numbers of STE family kinases.
A consecutive combination of both enrichment strategies therefore
allowed for the coverage of a larger part of the kinome than any one
technique alone. While kinobeads are very selective for kinases, the
ATP/ADP probes also enriched a large number of other nucleotide binding
proteins. Both methods were applied to the selectivity profiling of
the small molecular Aurora kinase inhibitor tozasertib in K562 cells.
Our data confirmed Aurora A, B, and BCR-ABL as the main targets of
tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar
off targets
MScDB: A Mass Spectrometry-centric Protein Sequence Database for Proteomics
Protein sequence databases are indispensable
tools for life science
research including mass spectrometry (MS)-based proteomics. In current
database construction processes, sequence similarity clustering is
used to reduce redundancies in the source data. Albeit powerful, it
ignores the peptide-centric nature of proteomic data and the fact
that MS is able to distinguish similar sequences. Therefore, we introduce
an approach that structures the protein sequence space at the peptide
level using theoretical and empirical information from large-scale
proteomic data to generate a mass spectrometry-centric protein sequence
database (MScDB). The core modules of MScDB are an <i>in-silico</i> proteolytic digest and a peptide-centric clustering algorithm that
groups protein sequences that are indistinguishable by mass spectrometry.
Analysis of various MScDB uses cases against five complex human proteomes,
resulting in 69 peptide identifications not present in UniProtKB as
well as 79 putative single amino acid polymorphisms. MScDB retains
∼99% of the identifications in comparison to common databases
despite a 3–48% increase in the theoretical peptide search
space (but comparable protein sequence space). In addition, MScDB
enables cross-species applications such as human/mouse graft models,
and our results suggest that the uncertainty in protein assignments
to one species can be smaller than 20%
Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity
Kinases
are involved in the regulation of many cellular processes
and aberrant kinase signaling has been implicated in human disease.
As a consequence, kinases are attractive drug targets. Assessing kinase
function and drug selectivity in a more physiological context is challenging
and often hampered by the generally low expression level of kinases
and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained
attention because they allow the profiling of hundreds of kinases
against one drug at the same time. Here, we directly compared two
such methods, notably, immobilized broad spectrum kinase inhibitors
(kinobeads) and active site labeling using desthiobiotin-ATP and -ADP
probes. Affinity purification of ∼100 kinases by either kinobeads
or ATP/ADP probes was readily achieved using 1 mg of cellular protein.
Bioinformatic analysis revealed a high degree of complementarity of
the two techniques. Kinobeads covered the Tyrosine Kinase family particularly
well and ATP probes enriched higher numbers of STE family kinases.
A consecutive combination of both enrichment strategies therefore
allowed for the coverage of a larger part of the kinome than any one
technique alone. While kinobeads are very selective for kinases, the
ATP/ADP probes also enriched a large number of other nucleotide binding
proteins. Both methods were applied to the selectivity profiling of
the small molecular Aurora kinase inhibitor tozasertib in K562 cells.
Our data confirmed Aurora A, B, and BCR-ABL as the main targets of
tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar
off targets
Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity
Kinases
are involved in the regulation of many cellular processes
and aberrant kinase signaling has been implicated in human disease.
As a consequence, kinases are attractive drug targets. Assessing kinase
function and drug selectivity in a more physiological context is challenging
and often hampered by the generally low expression level of kinases
and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained
attention because they allow the profiling of hundreds of kinases
against one drug at the same time. Here, we directly compared two
such methods, notably, immobilized broad spectrum kinase inhibitors
(kinobeads) and active site labeling using desthiobiotin-ATP and -ADP
probes. Affinity purification of ∼100 kinases by either kinobeads
or ATP/ADP probes was readily achieved using 1 mg of cellular protein.
Bioinformatic analysis revealed a high degree of complementarity of
the two techniques. Kinobeads covered the Tyrosine Kinase family particularly
well and ATP probes enriched higher numbers of STE family kinases.
A consecutive combination of both enrichment strategies therefore
allowed for the coverage of a larger part of the kinome than any one
technique alone. While kinobeads are very selective for kinases, the
ATP/ADP probes also enriched a large number of other nucleotide binding
proteins. Both methods were applied to the selectivity profiling of
the small molecular Aurora kinase inhibitor tozasertib in K562 cells.
Our data confirmed Aurora A, B, and BCR-ABL as the main targets of
tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar
off targets
MScDB: A Mass Spectrometry-centric Protein Sequence Database for Proteomics
Protein sequence databases are indispensable
tools for life science
research including mass spectrometry (MS)-based proteomics. In current
database construction processes, sequence similarity clustering is
used to reduce redundancies in the source data. Albeit powerful, it
ignores the peptide-centric nature of proteomic data and the fact
that MS is able to distinguish similar sequences. Therefore, we introduce
an approach that structures the protein sequence space at the peptide
level using theoretical and empirical information from large-scale
proteomic data to generate a mass spectrometry-centric protein sequence
database (MScDB). The core modules of MScDB are an <i>in-silico</i> proteolytic digest and a peptide-centric clustering algorithm that
groups protein sequences that are indistinguishable by mass spectrometry.
Analysis of various MScDB uses cases against five complex human proteomes,
resulting in 69 peptide identifications not present in UniProtKB as
well as 79 putative single amino acid polymorphisms. MScDB retains
∼99% of the identifications in comparison to common databases
despite a 3–48% increase in the theoretical peptide search
space (but comparable protein sequence space). In addition, MScDB
enables cross-species applications such as human/mouse graft models,
and our results suggest that the uncertainty in protein assignments
to one species can be smaller than 20%
MScDB: A Mass Spectrometry-centric Protein Sequence Database for Proteomics
Protein sequence databases are indispensable
tools for life science
research including mass spectrometry (MS)-based proteomics. In current
database construction processes, sequence similarity clustering is
used to reduce redundancies in the source data. Albeit powerful, it
ignores the peptide-centric nature of proteomic data and the fact
that MS is able to distinguish similar sequences. Therefore, we introduce
an approach that structures the protein sequence space at the peptide
level using theoretical and empirical information from large-scale
proteomic data to generate a mass spectrometry-centric protein sequence
database (MScDB). The core modules of MScDB are an <i>in-silico</i> proteolytic digest and a peptide-centric clustering algorithm that
groups protein sequences that are indistinguishable by mass spectrometry.
Analysis of various MScDB uses cases against five complex human proteomes,
resulting in 69 peptide identifications not present in UniProtKB as
well as 79 putative single amino acid polymorphisms. MScDB retains
∼99% of the identifications in comparison to common databases
despite a 3–48% increase in the theoretical peptide search
space (but comparable protein sequence space). In addition, MScDB
enables cross-species applications such as human/mouse graft models,
and our results suggest that the uncertainty in protein assignments
to one species can be smaller than 20%
Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity
Kinases
are involved in the regulation of many cellular processes
and aberrant kinase signaling has been implicated in human disease.
As a consequence, kinases are attractive drug targets. Assessing kinase
function and drug selectivity in a more physiological context is challenging
and often hampered by the generally low expression level of kinases
and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained
attention because they allow the profiling of hundreds of kinases
against one drug at the same time. Here, we directly compared two
such methods, notably, immobilized broad spectrum kinase inhibitors
(kinobeads) and active site labeling using desthiobiotin-ATP and -ADP
probes. Affinity purification of ∼100 kinases by either kinobeads
or ATP/ADP probes was readily achieved using 1 mg of cellular protein.
Bioinformatic analysis revealed a high degree of complementarity of
the two techniques. Kinobeads covered the Tyrosine Kinase family particularly
well and ATP probes enriched higher numbers of STE family kinases.
A consecutive combination of both enrichment strategies therefore
allowed for the coverage of a larger part of the kinome than any one
technique alone. While kinobeads are very selective for kinases, the
ATP/ADP probes also enriched a large number of other nucleotide binding
proteins. Both methods were applied to the selectivity profiling of
the small molecular Aurora kinase inhibitor tozasertib in K562 cells.
Our data confirmed Aurora A, B, and BCR-ABL as the main targets of
tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar
off targets
MScDB: A Mass Spectrometry-centric Protein Sequence Database for Proteomics
Protein sequence databases are indispensable
tools for life science
research including mass spectrometry (MS)-based proteomics. In current
database construction processes, sequence similarity clustering is
used to reduce redundancies in the source data. Albeit powerful, it
ignores the peptide-centric nature of proteomic data and the fact
that MS is able to distinguish similar sequences. Therefore, we introduce
an approach that structures the protein sequence space at the peptide
level using theoretical and empirical information from large-scale
proteomic data to generate a mass spectrometry-centric protein sequence
database (MScDB). The core modules of MScDB are an <i>in-silico</i> proteolytic digest and a peptide-centric clustering algorithm that
groups protein sequences that are indistinguishable by mass spectrometry.
Analysis of various MScDB uses cases against five complex human proteomes,
resulting in 69 peptide identifications not present in UniProtKB as
well as 79 putative single amino acid polymorphisms. MScDB retains
∼99% of the identifications in comparison to common databases
despite a 3–48% increase in the theoretical peptide search
space (but comparable protein sequence space). In addition, MScDB
enables cross-species applications such as human/mouse graft models,
and our results suggest that the uncertainty in protein assignments
to one species can be smaller than 20%
Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity
Kinases
are involved in the regulation of many cellular processes
and aberrant kinase signaling has been implicated in human disease.
As a consequence, kinases are attractive drug targets. Assessing kinase
function and drug selectivity in a more physiological context is challenging
and often hampered by the generally low expression level of kinases
and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained
attention because they allow the profiling of hundreds of kinases
against one drug at the same time. Here, we directly compared two
such methods, notably, immobilized broad spectrum kinase inhibitors
(kinobeads) and active site labeling using desthiobiotin-ATP and -ADP
probes. Affinity purification of ∼100 kinases by either kinobeads
or ATP/ADP probes was readily achieved using 1 mg of cellular protein.
Bioinformatic analysis revealed a high degree of complementarity of
the two techniques. Kinobeads covered the Tyrosine Kinase family particularly
well and ATP probes enriched higher numbers of STE family kinases.
A consecutive combination of both enrichment strategies therefore
allowed for the coverage of a larger part of the kinome than any one
technique alone. While kinobeads are very selective for kinases, the
ATP/ADP probes also enriched a large number of other nucleotide binding
proteins. Both methods were applied to the selectivity profiling of
the small molecular Aurora kinase inhibitor tozasertib in K562 cells.
Our data confirmed Aurora A, B, and BCR-ABL as the main targets of
tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar
off targets
MScDB: A Mass Spectrometry-centric Protein Sequence Database for Proteomics
Protein sequence databases are indispensable
tools for life science
research including mass spectrometry (MS)-based proteomics. In current
database construction processes, sequence similarity clustering is
used to reduce redundancies in the source data. Albeit powerful, it
ignores the peptide-centric nature of proteomic data and the fact
that MS is able to distinguish similar sequences. Therefore, we introduce
an approach that structures the protein sequence space at the peptide
level using theoretical and empirical information from large-scale
proteomic data to generate a mass spectrometry-centric protein sequence
database (MScDB). The core modules of MScDB are an <i>in-silico</i> proteolytic digest and a peptide-centric clustering algorithm that
groups protein sequences that are indistinguishable by mass spectrometry.
Analysis of various MScDB uses cases against five complex human proteomes,
resulting in 69 peptide identifications not present in UniProtKB as
well as 79 putative single amino acid polymorphisms. MScDB retains
∼99% of the identifications in comparison to common databases
despite a 3–48% increase in the theoretical peptide search
space (but comparable protein sequence space). In addition, MScDB
enables cross-species applications such as human/mouse graft models,
and our results suggest that the uncertainty in protein assignments
to one species can be smaller than 20%