107 research outputs found
5. 慢性円板状エリテマトーデスに続発せる棘細胞癌の1例について(第434(B)回千葉医学会例会 第15回千葉皮膚科臨床談話会)
Additional file 3: Table S3. Details of proteins identified in the study with reports on whether or not reported in PPD and gene ontology-based classification
Comprehensive Comparison of Collision Induced Dissociation and Electron Transfer Dissociation
Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues. However, unlike collision induced dissociation (CID), which has been studied for decades, the intricacies of ETD-based fragmentation have not yet been firmly established or systematically addressed. In this analysis, we have systematically compared the CID and ETD fragmentation patterns for the large majority of the peptides that do not contain such labile modifications. Using a standard 48 protein mix, we were able to measure false-positive rates for the experiments and also assess a large number of peptides for a detailed comparison of CID and ETD fragmentation pattern. Analysis of ∼19 000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in ∼20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%. Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted. We also present a novel strategy for automatic validation of peptide assignments based on identification of a peptide by consecutive CID and ETD fragmentation in an alternating mode
Analysis of Cellular Tyrosine Phosphorylation via Chemical Rescue of Conditionally Active Abl Kinase
Identifying
direct substrates targeted by protein kinases is important
in understanding cellular physiology and intracellular signal transduction.
Mass spectrometry-based quantitative proteomics provides a powerful
tool for comprehensively characterizing the downstream substrates
of protein kinases. This approach is efficiently applied to receptor
kinases that can be precisely, directly, and rapidly activated by
some agent, such as a growth factor. However, nonreceptor tyrosine
kinase Abl lacks the experimental advantage of extracellular
growth factors as immediate and direct stimuli. To circumvent this
limitation, we combine a chemical rescue approach with quantitative
phosphoproteomics to identify targets of Abl and their phosphorylation
sites with enhanced temporal resolution. Both known and novel putative
substrates are identified, presenting opportunities for studying unanticipated
functions of Abl under physiological and pathological conditions
Integrated Proteomic and Metabolic Analysis of Breast Cancer Progression
<div><p>One of the most persistent hallmarks of cancer biology is the preference of tumor cells to derive energy through glycolysis as opposed to the more efficient process of oxidative phosphorylation (OXPHOS). However, little is known about the molecular cascades by which oncogenic pathways bring about this metabolic switch. We carried out a quantitative proteomic and metabolic analysis of the MCF10A derived cell line model of breast cancer progression that includes parental cells and derivatives representing three different tumor grades of Ras-driven cancer with a common genetic background. A SILAC (Stable Isotope Labeling by Amino acids in Cell culture) labeling strategy was used to quantify protein expression in conjunction with subcellular fractionation to measure dynamic subcellular localization in the nucleus, cytosol and mitochondria. Protein expression and localization across cell lines were compared to cellular metabolic rates as a measure of oxidative phosphorylation (OXPHOS), glycolysis and cellular ATP. Investigation of the metabolic capacity of the four cell lines revealed that cellular OXPHOS decreased with breast cancer progression independently of mitochondrial copy number or electron transport chain protein expression. Furthermore, glycolytic lactate secretion did not increase in accordance with cancer progression and decreasing OXPHOS capacity. However, the relative expression and subcellular enrichment of enzymes critical to lactate and pyruvate metabolism supported the observed extracellular acidification profiles. This analysis of metabolic dysfunction in cancer progression integrated with global protein expression and subcellular localization is a novel and useful technique for determining organelle-specific roles of proteins in disease.</p> </div
Unbiased Discovery of Interactions at a Control Locus Driving Expression of the Cancer-Specific Therapeutic and Diagnostic Target, Mesothelin
Although significant effort is expended on identifying
transcripts/proteins that are up-regulated in cancer, there are few
reports on systematic elucidation of transcriptional mechanisms underlying
such druggable cancer-specific targets. The mesothelin (MSLN) gene
offers a promising subject, being expressed in a restricted pattern
normally, yet highly overexpressed in almost one-third of human malignancies
and a target of cancer immunotherapeutic trials. CanScript, a cis
promoter element, appears to control MSLN cancer-specific expression;
its related genomic sequences may up-regulate other cancer markers.
CanScript is a 20-nt bipartite element consisting of an SP1-like motif
and a consensus MCAT sequence. The latter recruits TEAD (TEA domain)
family members, which are universally expressed. Exploration of the
active CanScript element, especially the proteins binding to the SP1-like
motif, thus could reveal cancer-specific features having diagnostic
or therapeutic interest. The efficient identification of sequence-specific
DNA-binding proteins at a given locus, however, has lagged in biomarker
explorations. We used two orthogonal proteomics approachesunbiased
SILAC (stable isotope labeling by amino acids in cell culture)/DNA
affinity-capture/mass <u>s</u>pectrometry survey (SD-MS)
and a large transcription factor protein microarray (TFM)and
functional validation to explore systematically the CanScript interactome.
SD-MS produced nine candidates, and TFM, 18. The screens agreed in
confirming binding by TEAD proteins and by newly identified NAB1 and
NFATc. Among other identified candidates, we found functional roles
for ZNF24, NAB1 and RFX1 in MSLN expression by cancer cells. Combined
interactome screens yield an efficient, reproducible, sensitive, and
unbiased approach to identify sequence-specific DNA-binding proteins
and other participants in disease-specific DNA elements
Chromosome-centric Human Proteome Project: Deciphering Proteins Associated with Glioma and Neurodegenerative Disorders on Chromosome 12
In
line with the aims of the Chromosome-centric Human Proteome
Project (C-HPP) to completely annotate proteins of each chromosome
and biology/disease driven HPP (B/D-HPP) to decipher their relation
to diseases, we have generated a nonredundant catalogue of protein-coding
genes for Chromosome 12 (Chr. 12) and further annotated proteins associated
with major neurological disorders. Integrating high level proteomic
evidence from four major databases (neXtProt, Global Proteome Machine
(GPMdb), PeptideAtlas, and Human Protein Atlas (HPA)) along with Ensembl
data resource resulted in the identification of 1066 protein coding
genes, of which 171 were defined as “missing proteins”
based on the weak or complete absence of experimental evidence. With
functional annotations using DAVID and GAD, about 40% of the proteins
could be grouped as brain related with implications in cancer or neurological
disorders. We used published and unpublished high confidence mass
spectrometry data from our group and other literature consisting of
more than 5000 proteins derived from clinical specimens from patients
with human gliomas, Alzheimer’s disease, and Parkinson’s
disease and mapped it onto Chr. 12. We observed a total of 202 proteins
mapping to human Chr. 12, 136 of which were differentially expressed
in these disease conditions as compared to the normal. Functional
grouping indicated their association with cell cycle, cell-to-cell
signaling, and other important processes and networks, whereas their
disease association analysis confirmed neurological diseases and cancer
as the major group along with psycological disorders, with several
overexpressed genes/proteins mapping to 12q13-15 amplicon region.
Using multiple strategies and bioinformatics tools, we identified
103 differentially expressed proteins to have secretory potential,
17 of which have already been reported in direct analysis of the plasma
or cerebrospinal fluid (CSF) from the patients and 21 of them mapped
to cancer associated protein (CAPs) database that are amenable to
selective reaction monitoring (SRM) assays for targeted proteomic
analysis. Our analysis also reveals, for the first time, mass spectrometric
evidence for two “missing proteins” from Chr. 12, namely,
synaptic vesicle 2-related protein (SVOP) and IQ motif containing
D (IQCD). The analysis provides a snapshot of Chr. 12 encoded proteins
associated with gliomas and major neurological conditions and their
secretability which can be used to drive efforts for clinical applications
Summary of experimental workflow and data normalization.
<p>Lysates from 10A, T1K and CA1h cells were grown in light, medium or heavy SILAC medium, respectively, and equal numbers of cells were combined to obtain ratios of protein expression relative to parental cells. Light labeled CA1a cells were likewise combined with medium labeled T1K, and light to heavy ratios were normalized to the T1K fold change ratios in the 3-state experiment to calculate fold change of protein expression in CA1a cells relative to parental cells. After combining equal numbers of labeled cells, subcellular fractions of cytosolic, nuclear and mitochondrial proteins were obtained by centrifugation (500xg) over a sucrose gradient. Spectra from the peptide NPDDITQEEYGEFYK are shown to illustrate the fold change calculation for heat shock protein 90, which was identified in every cellular fraction and is disproportionately localized from the nucleus to the cytosol with breast cancer progression.</p
ATP generation relative to cell type and in response to the metabolic inhibitors, oligomycin alone, 2-DG alone, and the combination.
<p>Absolute ATP levels were quantified in each cell line. Each bar represents the mean of four independent measurements, and error bars are standard deviation of the mean. Significant differences were determined by comparing values for each cell line to 10A by Wilson’s T-Test (*p<0.05).</p
Cytosolic enrichment of glycolytic enzymes with breast cancer progression.
<p>Glycolytic enzymes (bold) are featured in the order by which they process intermediate molecules (italic) in the metabolic process of glycolysis. Protein expression in the cytosol is displayed as color-coded symbols to indicate fold-change relative to parental 10A cells.</p
Pharmacological profile of cellular rates of oxygen consumption and lactate production over time with the sequential injection (indicated by dashed lines) of the inhibitors (a) 1 μg/ml oligomycin, (b) 300nM FCCP, (c) 100mM 2-DG, and (d) 1 μM rotenone.
<p>A. Oxygen consumption rate (OCR) and B. extracellular acidification rate (ECAR) were measured in a Seahorse XF24 analyzer. Each data-point represents the mean of five independent samples measured in triplicate. Error bars indicate standard deviation of the mean.</p
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