4 research outputs found
An Approach for Triplex-Isobaric Peptide Termini Labeling (Triplex-IPTL)
Isobaric peptide termini labeling (IPTL) is based on
labeling of
both peptide termini with complementary isotopic labels resulting
in isobaric peptides. MS/MS analysis after IPTL derivatization produces
peptide-specific fragment ions which are distributed throughout the
MS/MS spectrum. Thus, several quantification points can be obtained
per peptide. In this report, we present triplex-IPTL, a chemical labeling
strategy for IPTL allowing the simultaneous quantification of three
states within one MS run. For this purpose, dimethylation of the N-terminal
amino group followed by dimethylation of lysines was used with different
stable isotopes of formaldehyde and cyanoborohydride. Upon LC-MS/MS
analysis, the combined samples revealed three corresponding isotopic
fragment ion series reflecting quantitatively the peptide ratios.
To support this multiplexing labeling strategy, we have further developed
the data analysis tool IsobariQ and included multidimensional VSN
normalization, statistical inference, and graphical visualization
of triplex-IPTL data and clustering of protein profiling patterns.
The power of the triplex-IPTL approach in combination with IsobariQ
was demonstrated through temporal profiling of HeLa cells incubated
with the kinesin Eg5 inhibitor S-Trityl-l-cysteine (STLC).
As a result, clusters of quantified proteins were found by their ratio
profiles which corresponded well to their gene ontology association
in mitotic arrest and cell death, respectively
An Approach for Triplex-Isobaric Peptide Termini Labeling (Triplex-IPTL)
Isobaric peptide termini labeling (IPTL) is based on
labeling of
both peptide termini with complementary isotopic labels resulting
in isobaric peptides. MS/MS analysis after IPTL derivatization produces
peptide-specific fragment ions which are distributed throughout the
MS/MS spectrum. Thus, several quantification points can be obtained
per peptide. In this report, we present triplex-IPTL, a chemical labeling
strategy for IPTL allowing the simultaneous quantification of three
states within one MS run. For this purpose, dimethylation of the N-terminal
amino group followed by dimethylation of lysines was used with different
stable isotopes of formaldehyde and cyanoborohydride. Upon LC-MS/MS
analysis, the combined samples revealed three corresponding isotopic
fragment ion series reflecting quantitatively the peptide ratios.
To support this multiplexing labeling strategy, we have further developed
the data analysis tool IsobariQ and included multidimensional VSN
normalization, statistical inference, and graphical visualization
of triplex-IPTL data and clustering of protein profiling patterns.
The power of the triplex-IPTL approach in combination with IsobariQ
was demonstrated through temporal profiling of HeLa cells incubated
with the kinesin Eg5 inhibitor S-Trityl-l-cysteine (STLC).
As a result, clusters of quantified proteins were found by their ratio
profiles which corresponded well to their gene ontology association
in mitotic arrest and cell death, respectively
Proximity Labeling Reveals Molecular Determinants of FGFR4 Endosomal Transport
The fibroblast growth factor receptors
(FGFRs) are important oncogenes
promoting tumor progression in many types of cancer, such as breast,
bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma.
However, little is known about how these receptors are internalized
and down-regulated in cells. We have here applied proximity biotin
labeling to identify proteins involved in FGFR4 signaling and trafficking.
For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal
tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed
in U2OS cells. Upon addition of biotin to these cells, proteins in
proximity to the FGFR4-BirA* fusion protein became biotinylated and
could be isolated and identified by quantitative mass spectrometry.
We identified in total 291 proteins, including 80 proteins that were
enriched in samples where the receptor was activated by the ligand
(FGF1), among them several proteins previously found to be involved
in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly,
many of the identified proteins were implicated in endosomal transport,
and by precise annotation we were able to trace the intracellular
pathways of activated FGFR4. Validating the data by confocal and three-dimensional
structured illumination microscopy analysis, we concluded that FGFR4
uses clathrin-mediated endocytosis for internalization and is further
sorted from early endosomes to the recycling compartment and the trans-Golgi
network. Depletion of cells for clathrin heavy chain led to accumulation
of FGFR4 at the cell surface and increased levels of active FGFR4
and PLCγ, while AKT and ERK signaling was diminished, demonstrating
that functional clathrin-mediated endocytosis is required for proper
FGFR4 signaling. Thus, this study reveals proteins and pathways involved
in FGFR4 transport and signaling that provide possible targets and
opportunities for therapeutic intervention in FGFR4 aberrant cancer
Proximity Labeling Reveals Molecular Determinants of FGFR4 Endosomal Transport
The fibroblast growth factor receptors
(FGFRs) are important oncogenes
promoting tumor progression in many types of cancer, such as breast,
bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma.
However, little is known about how these receptors are internalized
and down-regulated in cells. We have here applied proximity biotin
labeling to identify proteins involved in FGFR4 signaling and trafficking.
For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal
tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed
in U2OS cells. Upon addition of biotin to these cells, proteins in
proximity to the FGFR4-BirA* fusion protein became biotinylated and
could be isolated and identified by quantitative mass spectrometry.
We identified in total 291 proteins, including 80 proteins that were
enriched in samples where the receptor was activated by the ligand
(FGF1), among them several proteins previously found to be involved
in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly,
many of the identified proteins were implicated in endosomal transport,
and by precise annotation we were able to trace the intracellular
pathways of activated FGFR4. Validating the data by confocal and three-dimensional
structured illumination microscopy analysis, we concluded that FGFR4
uses clathrin-mediated endocytosis for internalization and is further
sorted from early endosomes to the recycling compartment and the trans-Golgi
network. Depletion of cells for clathrin heavy chain led to accumulation
of FGFR4 at the cell surface and increased levels of active FGFR4
and PLCγ, while AKT and ERK signaling was diminished, demonstrating
that functional clathrin-mediated endocytosis is required for proper
FGFR4 signaling. Thus, this study reveals proteins and pathways involved
in FGFR4 transport and signaling that provide possible targets and
opportunities for therapeutic intervention in FGFR4 aberrant cancer