32 research outputs found
Cell Death Proteomics Database: Consolidating Proteomics Data on Cell Death
Programmed cell death is a ubiquitous
process of utmost importance
for the development and maintenance of multicellular organisms. More
than 10 different types of programmed cell death forms have been discovered.
Several proteomics analyses have been performed to gain insight in
proteins involved in the different forms of programmed cell death.
To consolidate these studies, we have developed the cell death proteomics
(CDP) database, which comprehends data from apoptosis, autophagy,
cytotoxic granule-mediated cell death, excitotoxicity, mitotic catastrophe,
paraptosis, pyroptosis, and Wallerian degeneration. The CDP database
is available as a web-based database to compare protein identifications
and quantitative information across different experimental setups.
The proteomics data of 73 publications were integrated and unified
with protein annotations from UniProt-KB and gene ontology (GO). Currently,
more than 6,500 records of more than 3,700 proteins are included in
the CDP. Comparing apoptosis and autophagy using overrepresentation
analysis of GO terms, the majority of enriched processes were found
in both, but also some clear differences were perceived. Furthermore,
the analysis revealed differences and similarities of the proteome
between autophagosomal and overall autophagy. The CDP database represents
a useful tool to consolidate data from proteome analyses of programmed
cell death and is available at http://celldeathproteomics.uio.no
Quantitative Proteome Analysis Reveals RNA Processing Factors As Modulators of Ionizing Radiation-Induced Apoptosis in the <i>C. elegans</i> Germline.
The nematode <i>Caenorhabditis elegans</i> is an organism most recognized for forward and reverse
genetic and functional genomic approaches. Proteomic analyses of DNA
damage-induced apoptosis have not been shown because of a limited
number of cells undergoing apoptosis. We applied mass spectrometry-based
quantitative proteomics to evaluate protein changes induced by ionizing
radiation (IR) in isolated <i>C. elegans</i> germlines.
For this purpose, we used isobaric peptide termini labeling (IPTL)
combined with the data analysis tool IsobariQ, which utilizes MS/MS
spectra for relative quantification of peak pairs formed during fragmentation.
Using stringent statistical critera, we identified 48 proteins to
be significantly up- or down-regulated, most of which are part of
a highly interconnected proteinâprotein interaction network
dominated by proteins involved in translational control. RNA-mediated
depletion of a selection of the IR-regulated proteins revealed that
the conserved CAR-1/CGH-1/CEY-3 germline RNP complex acts as a novel
negative regulator of DNA-damage induced apoptosis. Finally, a central
role of nucleolar proteins in orchestrating these responses was confirmed
as the H/ACA snRNP protein GAR-1 was required for IR-induced apoptosis
in the <i>C. elegans</i> germline
Quantitative Proteome Analysis Reveals RNA Processing Factors As Modulators of Ionizing Radiation-Induced Apoptosis in the <i>C. elegans</i> Germline.
The nematode <i>Caenorhabditis elegans</i> is an organism most recognized for forward and reverse
genetic and functional genomic approaches. Proteomic analyses of DNA
damage-induced apoptosis have not been shown because of a limited
number of cells undergoing apoptosis. We applied mass spectrometry-based
quantitative proteomics to evaluate protein changes induced by ionizing
radiation (IR) in isolated <i>C. elegans</i> germlines.
For this purpose, we used isobaric peptide termini labeling (IPTL)
combined with the data analysis tool IsobariQ, which utilizes MS/MS
spectra for relative quantification of peak pairs formed during fragmentation.
Using stringent statistical critera, we identified 48 proteins to
be significantly up- or down-regulated, most of which are part of
a highly interconnected proteinâprotein interaction network
dominated by proteins involved in translational control. RNA-mediated
depletion of a selection of the IR-regulated proteins revealed that
the conserved CAR-1/CGH-1/CEY-3 germline RNP complex acts as a novel
negative regulator of DNA-damage induced apoptosis. Finally, a central
role of nucleolar proteins in orchestrating these responses was confirmed
as the H/ACA snRNP protein GAR-1 was required for IR-induced apoptosis
in the <i>C. elegans</i> germline
Quantitative Proteome Analysis Reveals RNA Processing Factors As Modulators of Ionizing Radiation-Induced Apoptosis in the <i>C. elegans</i> Germline.
The nematode <i>Caenorhabditis elegans</i> is an organism most recognized for forward and reverse
genetic and functional genomic approaches. Proteomic analyses of DNA
damage-induced apoptosis have not been shown because of a limited
number of cells undergoing apoptosis. We applied mass spectrometry-based
quantitative proteomics to evaluate protein changes induced by ionizing
radiation (IR) in isolated <i>C. elegans</i> germlines.
For this purpose, we used isobaric peptide termini labeling (IPTL)
combined with the data analysis tool IsobariQ, which utilizes MS/MS
spectra for relative quantification of peak pairs formed during fragmentation.
Using stringent statistical critera, we identified 48 proteins to
be significantly up- or down-regulated, most of which are part of
a highly interconnected proteinâprotein interaction network
dominated by proteins involved in translational control. RNA-mediated
depletion of a selection of the IR-regulated proteins revealed that
the conserved CAR-1/CGH-1/CEY-3 germline RNP complex acts as a novel
negative regulator of DNA-damage induced apoptosis. Finally, a central
role of nucleolar proteins in orchestrating these responses was confirmed
as the H/ACA snRNP protein GAR-1 was required for IR-induced apoptosis
in the <i>C. elegans</i> germline
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
Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches
Alternative
splicing is a mechanism in eukaryotes by which different
forms of mRNAs are generated from the same gene. Identification of
alternative splice variants requires the identification of peptides
specific for alternative splice forms. For this purpose, we generated
a human database that contains only unique tryptic peptides specific
for alternative splice forms from Swiss-Prot entries. Using this database
allows an easy access to splice variant-specific peptide sequences
that match to MS data. Furthermore, we combined this database without
alternative splice variant-1-specific peptides with human Swiss-Prot.
This combined database can be used as a general database for searching
of LCâMS data. LCâMS data derived from in-solution digests
of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies
were analyzed using these two databases. Several nonalternative splice
variant-1-specific peptides were found in both cell lines, and some
of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes
from Jurkat T cells revealed several nonalternative splice variant-1-specific
peptides, and some of them showed clear quantitative differences between
the two states
Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches
Alternative
splicing is a mechanism in eukaryotes by which different
forms of mRNAs are generated from the same gene. Identification of
alternative splice variants requires the identification of peptides
specific for alternative splice forms. For this purpose, we generated
a human database that contains only unique tryptic peptides specific
for alternative splice forms from Swiss-Prot entries. Using this database
allows an easy access to splice variant-specific peptide sequences
that match to MS data. Furthermore, we combined this database without
alternative splice variant-1-specific peptides with human Swiss-Prot.
This combined database can be used as a general database for searching
of LCâMS data. LCâMS data derived from in-solution digests
of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies
were analyzed using these two databases. Several nonalternative splice
variant-1-specific peptides were found in both cell lines, and some
of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes
from Jurkat T cells revealed several nonalternative splice variant-1-specific
peptides, and some of them showed clear quantitative differences between
the two states
Schematic view of the established method to enrich and analyze TG2 peptide substrates.
<p>A PTCEC digest of wheat gluten was mixed with a small amount of 5-BP which served as a substrate for TG2 in a transamidation reaction. The transamidated, biotinylated peptides were enriched from the digest using magnetic streptavidin beads, eluted with an excess of biotin and analyzed by LC-MS/MS. Database searching was performed using a database made up of all entries of <i>Triticum aestivum</i> present in the Uniprot database. In addition, MS/MS spectra were manually inspected.</p
TG2 peptide substrates identified by nano-LC MS/MS.
<p>Glutamine residues targeted by TG2 are given in bold.</p><p>DA, deamidation</p><p>The 9mer core region of T-cell epitopes are underlined.</p><p>âxâ indicates I or L.</p>a<p>number of protein entries in database.</p>b<p>not possible to determine which Q residue is transamidated (shown in bold and italic).</p>c<p>not possible to determine which Q residue is deamidated and which is transamidated.</p>d<p>not possible to determine whether Q2 or Q4 is targeted; however, this sequence has previously been shown to be targeted at Q4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014056#pone.0014056-Vader2" target="_blank">[17]</a>. The same Q residue is expected to be targeted in peptides #12â17.</p>e<p>identified in a sample incubated with TG2 for one minute.</p>f<p>two possible 9mer binding registers to HLA-DQ2.5.</p