16 research outputs found
Label-Free LC–MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis
Analysis
of the cerebrospinal fluid (CSF) proteome has proven valuable
to the study of neurodegenerative disorders. To identify new protein/pathway
alterations and candidate biomarkers for amyotrophic lateral sclerosis
(ALS), we performed comparative proteomic profiling of CSF from sporadic
ALS (sALS), healthy control (HC), and other neurological disease (OND)
subjects using label-free liquid chromatography-tandem mass spectrometry
(LC–MS/MS). A total of 1712 CSF proteins were detected and
relatively quantified by spectral counting. Levels of several proteins
with diverse biological functions were significantly altered in sALS
samples. Enrichment analysis was used to link these alterations to
biological pathways, which were predominantly related to inflammation,
neuronal activity, and extracellular matrix regulation. We then used
our CSF proteomic profiles to create a support vector machines classifier
capable of discriminating training set ALS from non-ALS (HC and OND)
samples. Four classifier proteins, WD repeat-containing protein 63,
amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule
3, were identified by feature selection and externally validated.
The resultant classifier distinguished ALS from non-ALS samples with
83% sensitivity and 100% specificity in an independent test set. Collectively,
our results illustrate the utility of CSF proteomic profiling for
identifying ALS protein/pathway alterations and candidate disease
biomarkers
Label-Free LC–MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis
Analysis
of the cerebrospinal fluid (CSF) proteome has proven valuable
to the study of neurodegenerative disorders. To identify new protein/pathway
alterations and candidate biomarkers for amyotrophic lateral sclerosis
(ALS), we performed comparative proteomic profiling of CSF from sporadic
ALS (sALS), healthy control (HC), and other neurological disease (OND)
subjects using label-free liquid chromatography-tandem mass spectrometry
(LC–MS/MS). A total of 1712 CSF proteins were detected and
relatively quantified by spectral counting. Levels of several proteins
with diverse biological functions were significantly altered in sALS
samples. Enrichment analysis was used to link these alterations to
biological pathways, which were predominantly related to inflammation,
neuronal activity, and extracellular matrix regulation. We then used
our CSF proteomic profiles to create a support vector machines classifier
capable of discriminating training set ALS from non-ALS (HC and OND)
samples. Four classifier proteins, WD repeat-containing protein 63,
amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule
3, were identified by feature selection and externally validated.
The resultant classifier distinguished ALS from non-ALS samples with
83% sensitivity and 100% specificity in an independent test set. Collectively,
our results illustrate the utility of CSF proteomic profiling for
identifying ALS protein/pathway alterations and candidate disease
biomarkers
Mitochondrial Proteomic Analysis of Cisplatin Resistance in Ovarian Cancer
Epithelial ovarian cancer (EOC) is the leading cause
of death among
women with gynecologic malignancies and accounts for approximately
6% of cancer deaths among women. Cisplatin and its analogues form
the backbone of the most active chemotherapy regimens in advanced
EOC; however, development of platinum resistance is common and typically
marks a transition in which curing the patient is no longer possible.
An emerging theme in many cancers is that mitochondrial dysfunction
contributes to an aggressive carcinogenic phenotype. We hypothesized
that changes in the mitochondrial proteome are required to support
development of cisplatin resistance in human EOC. To investigate this
hypothesis, an organellar proteomics approach was utilized to quantify
alterations in protein abundance in mitochondria enriched from isogenic
cisplatin-sensitive (A2780) and -resistant (A2780-CP20) human EOC
cells. Protein isolates from mitochondria-enriched fractions were
analyzed by high resolution liquid chromatography–tandem mass
spectrometry (LC–MS/MS), and relative abundance of identified
proteins was quantified by spectral counting. Pathway analyses revealed
significant increases in notch signaling pathways, cell survival,
and alternate apoptotic pathways in the A2780-CP20 subtype. Among
the alterations identified in the mitochondrial proteomic composition
in cisplatin-resistant EOC cells, activated leukocyte cell adhesion
molecule (AKAP12) and A kinase anchoring protein 12 (AKAP12) were
elevated, while nestin was diminished in the mitochondrial fraction
of A2780-CP20 relative to A2780. This was verified by immunoblot analysis.
These results confirm that important changes in the mitochondrial
proteome, many of which promote evasion of apoptosis and tumor invasiveness
and metastasis, are present in cisplatin-resistant EOC
Relative Quantitation of Proteins in Expressed Prostatic Secretion with a Stable Isotope Labeled Secretome Standard
Expressed prostatic secretion (EPS) is a proximal fluid directly derived from the prostate and, in the case of prostate cancer (PCa), is hypothesized to contain a repertoire of cancer-relevant proteins. Quantitative analysis of the EPS proteome may enable identification of proteins with utility for PCa diagnosis and prognosis. The present investigation demonstrates selective quantitation of proteins in EPS samples from PCa patients using a stable isotope labeled proteome standard (SILAP) generated through the selective harvest of the “secretome” from the PC3 prostate cancer cell line grown in stable isotope labeled cell culture medium. This stable isotope labeled secretome was digested with trypsin and equivalently added to each EPS digest, after which the resultant mixtures were analyzed by liquid chromatography–tandem mass spectrometry for peptide identification and quantification. Relative quantification of endogenous EPS peptides was accomplished by comparison of reconstructed mass chromatograms to those of the chemically identical SILAP peptides. A total of 86 proteins were quantified from 263 peptides in all of the EPS samples, 38 of which were found to be relevant to PCa. This work demonstrates the feasibility of using a SILAP secretome standard to simultaneously quantify many PCa-relevant proteins in EPS samples
Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes
ATM is a protein kinase that initiates a well-characterized
signaling cascade in cells exposed to ionizing radiation (IR). However,
the role for ATM in coordinating critical protein interactions and
subsequent exchanges within DNA damage response (DDR) complexes is
unknown. We combined SILAC-based tandem mass spectrometry and a subcellular
fractionation protocol to interrogate the proteome of irradiated cells
treated with or without the ATM kinase inhibitor KU55933. We developed
an integrative network analysis to identify and prioritize proteins
that were responsive to KU55933, specifically in chromatin, and that
were also enriched for physical interactions with known DNA repair
proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as
strong candidates. Using fluorescence recovery after photobleaching,
we found that the exchange of GFP-53BP1 in DDR complexes decreased
with KU55933. Further, we found that ANXA1 knockdown sensitized cells
to IR via a mechanism that was not potentiated by KU55933. Our study
reveals a role for ATM kinase activity in the dynamic exchange of
proteins in DDR complexes and identifies a role for ANXA1 in cellular
radioprotection
Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes
ATM is a protein kinase that initiates a well-characterized
signaling cascade in cells exposed to ionizing radiation (IR). However,
the role for ATM in coordinating critical protein interactions and
subsequent exchanges within DNA damage response (DDR) complexes is
unknown. We combined SILAC-based tandem mass spectrometry and a subcellular
fractionation protocol to interrogate the proteome of irradiated cells
treated with or without the ATM kinase inhibitor KU55933. We developed
an integrative network analysis to identify and prioritize proteins
that were responsive to KU55933, specifically in chromatin, and that
were also enriched for physical interactions with known DNA repair
proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as
strong candidates. Using fluorescence recovery after photobleaching,
we found that the exchange of GFP-53BP1 in DDR complexes decreased
with KU55933. Further, we found that ANXA1 knockdown sensitized cells
to IR via a mechanism that was not potentiated by KU55933. Our study
reveals a role for ATM kinase activity in the dynamic exchange of
proteins in DDR complexes and identifies a role for ANXA1 in cellular
radioprotection
Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes
ATM is a protein kinase that initiates a well-characterized
signaling cascade in cells exposed to ionizing radiation (IR). However,
the role for ATM in coordinating critical protein interactions and
subsequent exchanges within DNA damage response (DDR) complexes is
unknown. We combined SILAC-based tandem mass spectrometry and a subcellular
fractionation protocol to interrogate the proteome of irradiated cells
treated with or without the ATM kinase inhibitor KU55933. We developed
an integrative network analysis to identify and prioritize proteins
that were responsive to KU55933, specifically in chromatin, and that
were also enriched for physical interactions with known DNA repair
proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as
strong candidates. Using fluorescence recovery after photobleaching,
we found that the exchange of GFP-53BP1 in DDR complexes decreased
with KU55933. Further, we found that ANXA1 knockdown sensitized cells
to IR via a mechanism that was not potentiated by KU55933. Our study
reveals a role for ATM kinase activity in the dynamic exchange of
proteins in DDR complexes and identifies a role for ANXA1 in cellular
radioprotection
Elevated AKAP12 in Paclitaxel-Resistant Serous Ovarian Cancer Cells Is Prognostic and Predictive of Poor Survival in Patients
A majority
of high-grade (HG) serous ovarian cancer (SOC) patients
develop resistant disease despite high initial response rates to platinum/paclitaxel-based
chemotherapy. We identified shed/secreted proteins in preclinical
models of paclitaxel-resistant human HGSOC models and correlated these
candidate proteins with patient outcomes using public data from HGSOC
patients. Proteomic analyses of a HGSOC cell line secretome was compared
to those from a syngeneic paclitaxel-resistant variant and from a
line established from an intrinsically chemorefractory HGSOC patient.
Associations between the identified candidate proteins and patient
outcome were assessed in a discovery cohort of 545 patients and two
validation cohorts totaling 795 independent SOC patients. Among the
81 differentially abundant proteins identified (<i>q</i> < 0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes,
AKAP12 was verified to be elevated in all models of paclitaxel-resistant
HGSOC. Furthermore, elevated AKAP12 transcript expression was associated
with worse progression-free and overall survival. Associations with
outcome were observed in three independent cohorts and remained significant
after adjusted multivariate modeling. We further provide evidence
to support that differential gene methylation status is associated
with elevated expression of AKAP12 in taxol-resistant ovarian cancer
cells and ovarian cancer patient subsets. Elevated expression and
shedding/secretion of AKAP12 is characteristic of paclitaxel-resistant
HGSOC cells, and elevated AKAP12 transcript expression is a poor prognostic
and predictive marker for progression-free and overall survival in
SOC patients
HO-1, TACO-1, and HIF-1α expression in Elesclomol-treated melanoma cells.
<p>(<b>A</b>) Whole-cell (WC), (<b>B</b>) mitochondrial and WC, and (<b>C</b>) nuclear (Nu) and cytoplasmic (Cy) lysates, prepared from WM1158 metastatic melanoma cells following treatment with increasing doses of Elesclomol (ELM). Controls were WM1158 melanoma cells that received the drug vehicle DMSO, or no treatment (no tx). The blots were probed with antibody to HO-1, TACO-1, HIF-1α, or α-tubulin, which served as loading control. LDH5 was used as a cytoplasmic protein control.</p
Basal OCR in relation to the ECAR in short-term cultures of human melanocytes, and primary and metastatic melanoma cell lines.
<p>Depicted are HEMs (red symbol), primary (green symbol) and metastatic melanoma cell lines (blue symbol), and two Vemurafenib-resistant melanoma cell lines (dark blue symbol). The two melanoma cell lines derived from tumors of a same patient are depicted by open circles.</p