11 research outputs found
Lysine Methylation Mapping of Crenarchaeal DNA-Directed RNA Polymerases by Collision-Induced and Electron-Transfer Dissociation Mass Spectrometry
Enzymatic
machineries fundamental for information processing (e.g.,
transcription, replication, translation) in Archaea are simplified
versions of their eukaryotic counterparts. This is clearly noticeable
in the conservation of sequence and structure of corresponding enzymes
(see for example the archaeal DNA-directed RNA polymerase (RNAP)).
In Eukarya, post-translational modifications (PTMs) often serve as
functional regulatory factors for various enzymes and complexes. Among
the various PTMs, methylation and acetylation have been recently attracting
most attention. Nevertheless, little is known about such PTMs in Archaea,
and cross-methodological studies are scarce. We examined methylation
and N-terminal acetylation of endogenously purified crenarchaeal RNA
polymerase from <i>Sulfolobus shibatae</i> (<i>Ssh</i>) and <i>Sulfolobus acidocaldarius</i> (<i>Sac</i>). In-gel and in-solution protein digestion methods were combined
with collision-induced dissociation (CID) and electron-transfer dissociation
(ETD) mass spectrometry analysis. Overall, 20 and 26 methyl-lysines
for <i>S. shibatae</i> and <i>S. acidocaldarius</i> were identified, respectively. Furthermore, two N-terminal acetylation
sites for each of these organisms were assessed. As a result, we generated
a high-confidence data set for the mapping of methylation and acetylation
sites in both <i>Sulfolobus</i> species, allowing comparisons
with the data previously obtained for RNAP from <i>Sulfolobus
solfataricus</i> (<i>Sso</i>). We confirmed that all
observed methyl-lysines are on the surface of the RNAP
Human Basal Tear Peptidome Characterization by CID, HCD, and ETD Followed by in Silico and in Vitro Analyses for Antimicrobial Peptide Identification
Endogenous
peptides are valuable targets in the analysis of biological
processes. The tear film contains proteins and peptides released by
the tear duct mucosal cells, including antimicrobial peptides involved
in the protection against exogenous pathogens; however, the peptide
content of the tear liquid remains poorly characterized. We analyzed
naturally occurring peptides isolated from human basal tears. Mass
spectrometry analysis of endogenous peptides presents a number of
drawbacks, including size heterogeneity and nonpredictable fragmentation
patterns, among others. Therefore, CID, ETD, and HCD methods were
used for the characterization of the tear peptide content. The contribution
of DMSO as an additive of the chromatographic solvents was also evaluated.
We identified 157, 131, and 122 peptides using CID-, ETD-, and HCD-based
methods, respectively. Altogether, 234 different peptides were identified,
leading to the generation of the biggest data set of endogenous tear
peptides to date. The antimicrobial activity prediction analysis performed
in silico revealed different putative antimicrobial peptides. Two
of the extracellular glycoprotein lacritin peptides were de novo synthesized,
and their antimicrobial activity was confirmed in vitro. Our findings
demonstrate the benefits of using different fragmentation methods
for the analysis of endogenous peptides and provide a useful approach
for the discovery of peptides with antimicrobial activity
Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome
The
Spanish team of the Human Proteome Project (SpHPP) marked the
annotation of Chr16 and data analysis as one of its priorities. Precise
annotation of Chromosome 16 proteins according to C-HPP criteria is
presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of
DNA Elements (ENCODE) data sets were used to obtain further information
relative to cell/tissue specific chromosome 16 coding gene expression
patterns and to infer the presence of missing proteins. Twenty-four
shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant
experiments, representing 41% coverage of chromosome 16 proteins,
were performed. Furthermore, mapping of large-scale multicenter mass
spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines
into RNA-Seq data allowed further insights relative to correlation
of chromosome 16 transcripts and proteins. Detection and quantification
of chromosome 16 proteins in biological matrices by SRM procedures
are also primary goals of the SpHPP. Two strategies were undertaken:
one focused on known proteins, taking advantage of MS data already
available, and the second, aimed at the detection of the missing proteins,
is based on the expression of recombinant proteins to gather MS information
and optimize SRM methods that will be used in real biological samples.
SRM methods for 49 known proteins and for recombinant forms of 24
missing proteins are reported in this study
Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome
The
Spanish team of the Human Proteome Project (SpHPP) marked the
annotation of Chr16 and data analysis as one of its priorities. Precise
annotation of Chromosome 16 proteins according to C-HPP criteria is
presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of
DNA Elements (ENCODE) data sets were used to obtain further information
relative to cell/tissue specific chromosome 16 coding gene expression
patterns and to infer the presence of missing proteins. Twenty-four
shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant
experiments, representing 41% coverage of chromosome 16 proteins,
were performed. Furthermore, mapping of large-scale multicenter mass
spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines
into RNA-Seq data allowed further insights relative to correlation
of chromosome 16 transcripts and proteins. Detection and quantification
of chromosome 16 proteins in biological matrices by SRM procedures
are also primary goals of the SpHPP. Two strategies were undertaken:
one focused on known proteins, taking advantage of MS data already
available, and the second, aimed at the detection of the missing proteins,
is based on the expression of recombinant proteins to gather MS information
and optimize SRM methods that will be used in real biological samples.
SRM methods for 49 known proteins and for recombinant forms of 24
missing proteins are reported in this study
Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome
The
Spanish team of the Human Proteome Project (SpHPP) marked the
annotation of Chr16 and data analysis as one of its priorities. Precise
annotation of Chromosome 16 proteins according to C-HPP criteria is
presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of
DNA Elements (ENCODE) data sets were used to obtain further information
relative to cell/tissue specific chromosome 16 coding gene expression
patterns and to infer the presence of missing proteins. Twenty-four
shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant
experiments, representing 41% coverage of chromosome 16 proteins,
were performed. Furthermore, mapping of large-scale multicenter mass
spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines
into RNA-Seq data allowed further insights relative to correlation
of chromosome 16 transcripts and proteins. Detection and quantification
of chromosome 16 proteins in biological matrices by SRM procedures
are also primary goals of the SpHPP. Two strategies were undertaken:
one focused on known proteins, taking advantage of MS data already
available, and the second, aimed at the detection of the missing proteins,
is based on the expression of recombinant proteins to gather MS information
and optimize SRM methods that will be used in real biological samples.
SRM methods for 49 known proteins and for recombinant forms of 24
missing proteins are reported in this study
Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome
The
Spanish team of the Human Proteome Project (SpHPP) marked the
annotation of Chr16 and data analysis as one of its priorities. Precise
annotation of Chromosome 16 proteins according to C-HPP criteria is
presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of
DNA Elements (ENCODE) data sets were used to obtain further information
relative to cell/tissue specific chromosome 16 coding gene expression
patterns and to infer the presence of missing proteins. Twenty-four
shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant
experiments, representing 41% coverage of chromosome 16 proteins,
were performed. Furthermore, mapping of large-scale multicenter mass
spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines
into RNA-Seq data allowed further insights relative to correlation
of chromosome 16 transcripts and proteins. Detection and quantification
of chromosome 16 proteins in biological matrices by SRM procedures
are also primary goals of the SpHPP. Two strategies were undertaken:
one focused on known proteins, taking advantage of MS data already
available, and the second, aimed at the detection of the missing proteins,
is based on the expression of recombinant proteins to gather MS information
and optimize SRM methods that will be used in real biological samples.
SRM methods for 49 known proteins and for recombinant forms of 24
missing proteins are reported in this study
Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome
The
Spanish team of the Human Proteome Project (SpHPP) marked the
annotation of Chr16 and data analysis as one of its priorities. Precise
annotation of Chromosome 16 proteins according to C-HPP criteria is
presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of
DNA Elements (ENCODE) data sets were used to obtain further information
relative to cell/tissue specific chromosome 16 coding gene expression
patterns and to infer the presence of missing proteins. Twenty-four
shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant
experiments, representing 41% coverage of chromosome 16 proteins,
were performed. Furthermore, mapping of large-scale multicenter mass
spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines
into RNA-Seq data allowed further insights relative to correlation
of chromosome 16 transcripts and proteins. Detection and quantification
of chromosome 16 proteins in biological matrices by SRM procedures
are also primary goals of the SpHPP. Two strategies were undertaken:
one focused on known proteins, taking advantage of MS data already
available, and the second, aimed at the detection of the missing proteins,
is based on the expression of recombinant proteins to gather MS information
and optimize SRM methods that will be used in real biological samples.
SRM methods for 49 known proteins and for recombinant forms of 24
missing proteins are reported in this study
Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome
The
Spanish team of the Human Proteome Project (SpHPP) marked the
annotation of Chr16 and data analysis as one of its priorities. Precise
annotation of Chromosome 16 proteins according to C-HPP criteria is
presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of
DNA Elements (ENCODE) data sets were used to obtain further information
relative to cell/tissue specific chromosome 16 coding gene expression
patterns and to infer the presence of missing proteins. Twenty-four
shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant
experiments, representing 41% coverage of chromosome 16 proteins,
were performed. Furthermore, mapping of large-scale multicenter mass
spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines
into RNA-Seq data allowed further insights relative to correlation
of chromosome 16 transcripts and proteins. Detection and quantification
of chromosome 16 proteins in biological matrices by SRM procedures
are also primary goals of the SpHPP. Two strategies were undertaken:
one focused on known proteins, taking advantage of MS data already
available, and the second, aimed at the detection of the missing proteins,
is based on the expression of recombinant proteins to gather MS information
and optimize SRM methods that will be used in real biological samples.
SRM methods for 49 known proteins and for recombinant forms of 24
missing proteins are reported in this study
Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome
The
Spanish team of the Human Proteome Project (SpHPP) marked the
annotation of Chr16 and data analysis as one of its priorities. Precise
annotation of Chromosome 16 proteins according to C-HPP criteria is
presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of
DNA Elements (ENCODE) data sets were used to obtain further information
relative to cell/tissue specific chromosome 16 coding gene expression
patterns and to infer the presence of missing proteins. Twenty-four
shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant
experiments, representing 41% coverage of chromosome 16 proteins,
were performed. Furthermore, mapping of large-scale multicenter mass
spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines
into RNA-Seq data allowed further insights relative to correlation
of chromosome 16 transcripts and proteins. Detection and quantification
of chromosome 16 proteins in biological matrices by SRM procedures
are also primary goals of the SpHPP. Two strategies were undertaken:
one focused on known proteins, taking advantage of MS data already
available, and the second, aimed at the detection of the missing proteins,
is based on the expression of recombinant proteins to gather MS information
and optimize SRM methods that will be used in real biological samples.
SRM methods for 49 known proteins and for recombinant forms of 24
missing proteins are reported in this study
Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome
The
Spanish team of the Human Proteome Project (SpHPP) marked the
annotation of Chr16 and data analysis as one of its priorities. Precise
annotation of Chromosome 16 proteins according to C-HPP criteria is
presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of
DNA Elements (ENCODE) data sets were used to obtain further information
relative to cell/tissue specific chromosome 16 coding gene expression
patterns and to infer the presence of missing proteins. Twenty-four
shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant
experiments, representing 41% coverage of chromosome 16 proteins,
were performed. Furthermore, mapping of large-scale multicenter mass
spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines
into RNA-Seq data allowed further insights relative to correlation
of chromosome 16 transcripts and proteins. Detection and quantification
of chromosome 16 proteins in biological matrices by SRM procedures
are also primary goals of the SpHPP. Two strategies were undertaken:
one focused on known proteins, taking advantage of MS data already
available, and the second, aimed at the detection of the missing proteins,
is based on the expression of recombinant proteins to gather MS information
and optimize SRM methods that will be used in real biological samples.
SRM methods for 49 known proteins and for recombinant forms of 24
missing proteins are reported in this study