John Asara
Beth Israel Deaconess Medical Center, Medicine, Faculty Member
Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an... more
Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.
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Research Interests: Cancer()
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Organ wasting, related to changes in nutrition and metabolic activity of cells and tissues, is observed under conditions of starvation and in the context of diseases, including cancers. We have developed a model for organ wasting in adult... more
Organ wasting, related to changes in nutrition and metabolic activity of cells and tissues, is observed under conditions of starvation and in the context of diseases, including cancers. We have developed a model for organ wasting in adult Drosophila, whereby overproliferation induced by activation of Yorkie, the Yap1 oncogene ortholog, in intestinal stem cells leads to wasting of the ovary, fat body, and muscle. These organ-wasting phenotypes are associated with a reduction in systemic insulin/IGF signaling due to increased expression of the secreted insulin/IGF antagonist ImpL2 from the overproliferating gut. Strikingly, expression of rate-limiting glycolytic enzymes and central components of the insulin/IGF pathway is upregulated with activation of Yorkie in the gut, which may provide a mechanism for this overproliferating tissue to evade the effect of ImpL2. Altogether, our study provides insights into the mechanisms underlying organ-wasting phenotypes in Drosophila and how overproliferating tissues adapt to global changes in metabolism.
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Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used to analyze oligonucleotides. However, success has been limited by cation adduction and high detection limits. Both of these problems are due to the... more
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used to analyze oligonucleotides. However, success has been limited by cation adduction and high detection limits. Both of these problems are due to the high net negative charge that oligonucleotides carry on the phosphodiester backbone. Comatrixes such as ammonium salts with UV absorbers such as 3-hydroxypicolinic acid, 2,4,6-trihydroxyacetophenone, and 6-aza-2-thiothymine have been used to improve the spectral quality for oligonucleotides in MALDI MS. Organic bases have also been used as co-matrixes; however, the most popular matrix, 3-hydroxypicolinic acid, is not compatible with these additives. We have found that the tetraamine spermine as a matrix additive can successfully eliminate cation adduction and lower the detection limits for DNA in the MALDI experiment, without having to resort to desalting steps. The results suggest that multiply protonated spermine molecules function better than ammonium ions in neutralizing oligonucleotides and displacing alkali cations. Protonated spermine is chemically similar to ammonium ions since it binds to the phosphate backbone and releases protons to the phosphate groups. Spermine can be used successfully with the matrixes 6-aza-2-thiothymine and 80% anthranilic acid/20% nicotinic acid but not with 3-hydroxypicolinic acid. The additive also works well for the analysis of metalated DNA.
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Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated... more
Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription
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Hippo signaling is a tumor-suppressor pathway involved in organ size control and tumorigenesis through the inhibition of YAP and TAZ. Here, we show that energy stress induces YAP cytoplasmic retention and S127 phosphorylation and inhibits... more
Hippo signaling is a tumor-suppressor pathway involved in organ size control and tumorigenesis through the inhibition of YAP and TAZ. Here, we show that energy stress induces YAP cytoplasmic retention and S127 phosphorylation and inhibits YAP transcriptional activity and YAP-dependent transformation. These effects require the central metabolic sensor AMP-activated protein kinase (AMPK) and the upstream Hippo pathway components Lats1/Lats2 and angiomotin-like 1 (AMOTL1). Furthermore, we show that AMPK directly phosphorylates S793 of AMOTL1. AMPK activation stabilizes and increases AMOTL1 steady-state protein levels, contributing to YAP inhibition. The phosphorylation-deficient S793Ala mutant of AMOTL1 showed a shorter half-life and conferred resistance to energy-stress-induced YAP inhibition. Our findings link energy sensing to the Hippo-YAP pathway and suggest that YAP may integrate spatial (contact inhibition), mechanical, and metabolic signals to control cellular proliferation and...
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Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells, but little is known as to what metabolic changes benefit breast... more
Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells, but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the nonoxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBP) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis a...
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Deficiency in repair of damaged DNA leads to genomic instability and is closely associated with tumorigenesis. Most DNA double-strand-breaks (DSBs) are repaired by two major mechanisms, homologous-recombination (HR) and... more
Deficiency in repair of damaged DNA leads to genomic instability and is closely associated with tumorigenesis. Most DNA double-strand-breaks (DSBs) are repaired by two major mechanisms, homologous-recombination (HR) and non-homologous-end-joining (NHEJ). Although Akt has been reported to suppress HR, its role in NHEJ remains elusive. Here, we report that Akt phosphorylates XLF at Thr181 to trigger its dissociation from the DNA ligase IV/XRCC4 complex, and promotes its interaction with 14-3-3β leading to XLF cytoplasmic retention, where cytosolic XLF is subsequently degraded by SCF(β-TRCP) in a CKI-dependent manner. Physiologically, upon DNA damage, XLF-T181E expressing cells display impaired NHEJ and elevated cell death. Whereas a cancer-patient-derived XLF-R178Q mutant, deficient in XLF-T181 phosphorylation, exhibits an elevated tolerance of DNA damage. Together, our results reveal a pivotal role for Akt in suppressing NHEJ and highlight the tight connection between aberrant Akt hyper-activation and deficiency in timely DSB repair, leading to genomic instability and tumorigenesis.
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BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this... more
BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated
Research Interests: Genetics, Mass Spectrometry, Gene expression, Signal Transduction, Biological Sciences, and 13 morePlant Physiology, Protein Kinases, Plant, Growth and development, Threonine, Phosphorylation, Arabidopsis, Phytosterols, Amino Acid Sequence, Mutants, Recombinant Proteins, Receptor-Like Kinase, and Fresh Weight
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Elevated glycolysis is a common metabolic trait of cancer, but what drives such metabolic reprogramming remains incompletely clear. We report here a novel transcriptional repressor-mediated negative regulation of glycolysis. ZBTB7A, a... more
Elevated glycolysis is a common metabolic trait of cancer, but what drives such metabolic reprogramming remains incompletely clear. We report here a novel transcriptional repressor-mediated negative regulation of glycolysis. ZBTB7A, a member of the POK (POZ/BTB and Krüppel) transcription repressor family, directly binds to the promoter and represses the transcription of critical glycolytic genes, including GLUT3, PFKP, and PKM. Analysis of The Cancer Genome Atlas (TCGA) data sets reveals that the ZBTB7A locus is frequently deleted in many human tumors. Significantly, reduced ZBTB7A expression correlates with up-regulation of the glycolytic genes and poor survival in colon cancer patients. Remarkably, while ZBTB7A-deficient tumors progress exceedingly fast, they exhibit an unusually heightened sensitivity to glycolysis inhibition. Our study uncovers a novel tumor suppressor role of ZBTB7A in directly suppressing glycolysis.
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Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is... more
Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is present in vivo and in vitro. In vivo, tear samples were collected from age-matched controls with no eye disease (n = 36) and KC diagnosed subjects (n = 17). Samples were processed for proteomics using LC-MS/MS. In vitro, cells were isolated from controls (Human Corneal Fibroblasts-HCF) and KC subjects (Human Keratoconus Cells-HKC) and stimulated with a Vitamin C (VitC) derivative for 4 weeks, and with one of the three transforming growth factor-beta (TGF-β) isoforms. Samples were analyzed using real-time PCR and Western Blots. By using proteomics analysis, the Gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP) was found to be the best independent biomarker able to discriminate between KC and controls. The intensity of GCDFP-15/PIP was significantly higher in healthy subjects compared to KC-diagnosed. Similar findings were seen in vitro, using a 3D culture model. All three TGF-β isoforms significantly down-regulated the expression of GCDFP-15/PIP. Zinc-alpha-2-glycoprotein (AZGP1), a protein that binds to PIP, was identified by proteomics and cell culture to be highly regulated. In this study by different complementary techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for KC disease. It is likely that exploring the GCDFP-15/PIP-AZGP1 interactions will help better understand the mechanism of KC disease.
Research Interests: Biomarkers, Vitamins, Proteomics, Cornea, Gene expression, and 14 moreMultidisciplinary, Western blotting, Humans, Keratoconus, Young Adult, Tandem Mass Spectrometry, Glycoproteins, PLoS one, High Performance Liquid Chromatography, Ascorbic Acid, Adult, Transforming Growth Factor Beta, Human Fibroblasts, and Proteome
Prostate cancer is the most prevalent cancer in males, and treatment options are limited for advanced forms of the disease. Loss of the PTEN and TP53 tumor suppressor genes is commonly observed in prostate cancer, whereas their compound... more
Prostate cancer is the most prevalent cancer in males, and treatment options are limited for advanced forms of the disease. Loss of the PTEN and TP53 tumor suppressor genes is commonly observed in prostate cancer, whereas their compound loss is often observed in advanced prostate cancer. Here, we show that PARP inhibition triggers a p53-dependent cellular senescence in a PTEN-deficient setting in the prostate. Surprisingly, we also find that PARP-induced cellular senescence is morphed into an apoptotic response upon compound loss of PTEN and p53. We further show that superactivation of the prosurvival PI3K-AKT signaling pathway limits the efficacy of a PARP single-agent treatment, and that PARP and PI3K inhibitors effectively synergize to suppress tumorigenesis in human prostate cancer cell lines and in a Pten/Trp53-deficient mouse model of advanced prostate cancer. Our findings, therefore, identify a combinatorial treatment with PARP and PI3K inhibitors as an effective option for P...
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Research Interests: Immunohistochemistry, Transmission Electron Microscopy, Mitochondria, Transcription Factors, Western blotting, and 13 moreBiological Sciences, RNA interference, Humans, Female, Animals, Male, Middle Aged, Neoplasm Invasiveness, Oxidative phosphorylation, Neoplasms, Gene Expression Analysis, Oxygen Consumption, and Gene expression profiling
Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1).... more
Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating...
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Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for... more
Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for recellularization with selected cell types. We aimed to up-scale this process, generating biocompatible scaffolds of a clinically relevant scale. Rat, porcine, and human lungs were decellularized by detergent perfusion at constant pressures. Collagen, elastin, and glycosaminoglycan content of scaffolds were quantified by colorimetric assays. Proteomic analysis was performed by microcapillary liquid chromatography tandem mass spectrometry. Extracellular matrix (ECM) slices were cultured with human umbilical vein endothelial cells (HUVEC), small airway epithelial cells (SAEC), or pulmonary alveolar epithelial cells (PAECs) and evaluated by time-lapse live cell microscopy and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Whole-orga...
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No comprehensive metabolic profile of trait anxiety is to date available. To identify metabolic biosignatures for different anxiety states, we compared mice selectively inbred for ∼ 40 generations for high (HAB), normal (NAB) or low (LAB)... more
No comprehensive metabolic profile of trait anxiety is to date available. To identify metabolic biosignatures for different anxiety states, we compared mice selectively inbred for ∼ 40 generations for high (HAB), normal (NAB) or low (LAB) anxiety-related behavior. Using a mass spectrometry-based targeted metabolomics approach, we quantified the levels of 257 unique metabolites in the cingulate cortex and plasma of HAB, NAB and LAB mice. We then pinpointed affected molecular systems in anxiety-related behavior by an in silico pathway and network prediction analysis followed by validation of in silico predicted alterations with molecular assays. We found distinct metabolic profiles for different trait anxiety states and detected metabolites with altered levels both in cingulate cortex and plasma. Metabolomics data revealed common candidate biomarkers in cingulate cortex and plasma for anxiety traits and in silico pathway analysis implicated amino acid metabolism, pyruvate metabolism, oxidative stress and apoptosis in the regulation of anxiety-related behavior. We report characteristic biosignatures for trait anxiety states and provide a network map of pathways involved in anxiety-related behavior. Pharmacological targeting of these pathways will enable a mechanism-based approach for identifying novel therapeutic targets for anxiety disorders.
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We performed multiple analyses of Tyrannosaurus rex (specimen MOR 1125) fibrous cortical and medullary tissues remaining after demineralization. The results indicate that collagen I, the main organic component of bone, has been preserved... more
We performed multiple analyses of Tyrannosaurus rex (specimen MOR 1125) fibrous cortical and medullary tissues remaining after demineralization. The results indicate that collagen I, the main organic component of bone, has been preserved in low concentrations in these tissues. The findings were independently confirmed by mass spectrometry. We propose a possible chemical pathway that may contribute to this preservation. The presence of endogenous protein in dinosaur bone may validate hypotheses about evolutionary relationships, rates, and patterns of molecular change and degradation, as well as the chemical stability of molecules over time.
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Research Interests: Kinetics, Mass Spectrometry, Cancer, Science, Biology, and 20 moreMedicine, Multidisciplinary, Cell line, Humans, Glucose, Pyruvate Kinase, Mice, Female, Animals, Male, Phosphorylation, Glycolysis, Enzyme, Isoenzymes, Neoplasms, Enzyme activity, Cell Proliferation, Recombinant Proteins, Histidine, and Adenosine Triphosphate
Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified... more
Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and Caenorhabditis elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.
Research Interests: Caenorhabditis elegans, Science, Autophagy, Transcription Factors, Multidisciplinary, and 15 moreEnergy Metabolism, Signal Transduction, Cell line, Humans, Metformin, Insulin, Liver, Mice, Animals, Phosphorylation, Hepatocytes, Environmental Stress, Nutrient Availability, Protein Kinase, and Cell Survival
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Research Interests: Prostate Cancer, Gene expression, Multidisciplinary, Gene Silencing, Signal Transduction, and 14 moreCell line, Humans, Male, Gene Regulatory Networks, Transcription Factor, PLoS one, Western blot, Protein Expression, Reproducibility of Results, Transfection, Metabolic pathway, Metabolome, Gene expression profiling, and Down-Regulation
Research Interests: Data Analysis, Mass Spectrometry, Biological Sciences, Stable Isotope, Peptides, and 8 moreSodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, High Performance Liquid Chromatography, CHEMICAL SCIENCES, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Relative Abundance, Gel electrophoresis, Isotope Labeling, and Region of Interest
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Research Interests: Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology, Genetics, and 61 moreMarine Biology, Neuroscience, Environmental Science, Geophysics, Physics, Materials Science, Quantum Physics, Developmental Biology, Immunology, Climate Change, Molecular Biology, Structural Biology, Genomics, RNA, Metabolism, Computational Biology, Transcriptomics, Catalysis, Biotechnology, Systems Biology, Cancer, Biology, Metabolomics, Cell Cycle, Proteomics, Ecology, Drug Discovery, Evolution, Regulation, Nanotechnology, Astrophysics, Neurobiology, Medicine, Multidisciplinary, Palaeobiology, Functional Genomics, Nature, Signal Transduction, Astronomy, DNA, Cell line, Humans, Pyruvate Kinase, Animals, Cell Signalling, Medical Research, Phosphorylation, Glycolysis, Enzyme, Phosphotyrosine, Cells, Energy Production, Lysine, HeLa cells, Substrate Specificity, Cell Proliferation, Growth Factor, Protein Binding, Enzymatic Activity, Earth Science, and Tyrosine Phosphorylation(Marine Biology, Neuroscience, Environmental Science, Geophysics, Physics, Materials Science, Quantum Physics, Developmental Biology, Immunology, Climate Change, Molecular Biology, Structural Biology, Genomics, RNA, Metabolism, Computational Biology, Transcriptomics, Catalysis, Biotechnology, Systems Biology, Cancer, Biology, Metabolomics, Cell Cycle, Proteomics, Ecology, Drug Discovery, Evolution, Regulation, Nanotechnology, Astrophysics, Neurobiology, Medicine, Multidisciplinary, Palaeobiology, Functional Genomics, Nature, Signal Transduction, Astronomy, DNA, Cell line, Humans, Pyruvate Kinase, Animals, Cell Signalling, Medical Research, Phosphorylation, Glycolysis, Enzyme, Phosphotyrosine, Cells, Energy Production, Lysine, HeLa cells, Substrate Specificity, Cell Proliferation, Growth Factor, Protein Binding, Enzymatic Activity, Earth Science, and Tyrosine Phosphorylation)
(Marine Biology, Neuroscience, Environmental Science, Geophysics, Physics, Materials Science, Quantum Physics, Developmental Biology, Immunology, Climate Change, Molecular Biology, Structural Biology, Genomics, RNA, Metabolism, Computational Biology, Transcriptomics, Catalysis, Biotechnology, Systems Biology, Cancer, Biology, Metabolomics, Cell Cycle, Proteomics, Ecology, Drug Discovery, Evolution, Regulation, Nanotechnology, Astrophysics, Neurobiology, Medicine, Multidisciplinary, Palaeobiology, Functional Genomics, Nature, Signal Transduction, Astronomy, DNA, Cell line, Humans, Pyruvate Kinase, Animals, Cell Signalling, Medical Research, Phosphorylation, Glycolysis, Enzyme, Phosphotyrosine, Cells, Energy Production, Lysine, HeLa cells, Substrate Specificity, Cell Proliferation, Growth Factor, Protein Binding, Enzymatic Activity, Earth Science, and Tyrosine Phosphorylation)
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Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D... more
Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.
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Research Interests: Evolutionary Biology, Genetics, Molecular Evolution, Mass Spectrometry, Scanning Electron Microscopy, and 12 moreImmunohistochemistry, Molecular, Fossil record, Elephants, Fossils, Animals, Tandem Mass Spectrometry, Western blot, Amino Acid Profile, Amino Acid Sequence, Biochemistry and cell biology, and Nucleic Acid
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Proteomic profiling using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) enables the identification of biomarkers for cancer. We evaluated the sensitivity and specificity of SELDI-TOF MS for... more
Proteomic profiling using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) enables the identification of biomarkers for cancer. We evaluated the sensitivity and specificity of SELDI-TOF MS for detection of established hepatocellular cancer (HCC) and compared it against alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive AFP (AFP-L3), and prothrombin induced by vitamin K absence-II (PIVKA-II). Forty-one patients with HCC and 51 patients with hepatitis C cirrhosis were enrolled. Serum was analyzed by SELDI-TOF MS using three Ciphergen protein array types. An 11-peak algorithm for HCC detection was identified. Using the AFP cutoff of 20 ng/mL, the sensitivity was 73% and the specificity was 71%. Using the AFP-L3 cutoff of 10% yielded a sensitivity of 63% and a specificity of 94%. Using the PIVKA-II cutoff of 125 milliabsorbance units (mAU), the sensitivity was 84% and the specificity was 69%. Overall, the sensitivity and specificity of SELDI-TOF MS for HCC were 79% and 86%, respectively. In multivariate analysis, the 11-peak SELDI profile was predictive of HCC independent of AFP, PIVKA, and AFP-L3. Among eight patients with the largest tumor size of <2 cm, SELDI-TOF MS correctly identified seven whereas AFP, AFP-L3, and PIVKA-II identified only three, one, and one, respectively. One of the 11 peaks in the SELDI-TOF MS 11-peak predictor from SELDI-TOF MS was identified as cystatin C. SELDI-TOF MS accurately distinguished patients with HCC from those with hepatitis C virus cirrhosis, was more accurate than traditional biomarkers in identifying small tumors, and should be further evaluated.