Interactome Profiling of DNA Damage Response (DDR) Mediators with Immunoprecipitation-Mass Spectrometry.

Immunoprecipitation-mass spectrometry (IP-MS) is a versatile tool to probe for global protein-protein interactions (PPIs) in biological samples. Such interactions coordinate complex biological processes, such as the DNA damage response (DDR). Induction of DNA damage activates signaling networks where posttranslational modifications cause PPI that facilitate DNA repair and cell cycle coordination.

Combined Alcohol Exposure and KRAS Mutation in Human Pancreatic Ductal Epithelial Cells Induces Proliferation and Alters Subtype Signatures Determined by Multi-Omics Analysis.

Pancreatic Ductal adenocarcinoma (PDAC) is an aggressive cancer commonly exhibiting KRAS-activating mutations. Alcohol contributes to the risk of developing PDAC in humans, and murine models have shown alcohol consumption in the context of KRAS mutation in the pancreas promotes the development of PDAC. The molecular signatures in pancreas cells altered by alcohol exposure in the context of mutant KRAS could identify pathways related to the etiology of PDAC.

An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry.

This protocol represents an optimized proteomics-based protocol for the endogenous protein enrichment and protein-protein interaction analysis. This 2-step protocol consists of: 1) co-immunoprecipitation of the bait protein; 2) the bait-protein interactions analysis using LC-MS/MS. Here, we used Dynabeads® for the enrichment of the target protein (the bait) and its interactors.

An audit of the effect of SGLT2 inhibitor cessation in a pre-admission clinic before and after Australian Diabetes Society guidelines.

Perioperative ketoacidosis is an important adverse event related to the use of sodium glucose co-transporter 2 inhibitor (SGLT2i). We compared perioperative outcomes in patients on SGLT2i, before and after protocolised pre-operative cessation of the drug. There were no cases of clinically detected diabetic ketoacidosis in the 96 patients included in the study.

Aminopyrazole based CDK9 PROTAC sensitizes pancreatic cancer cells to venetoclax.

Cyclin-dependent kinase 9 (CDK9) is a member of the cyclin-dependent kinase (CDK) family which is involved in transcriptional regulation of several genes, including the oncogene Myc, and is a validated target for pancreatic cancer. Here we report the development of an aminopyrazole based proteolysis targeting chimera (PROTAC 2) that selectively degrades CDK9 (DC = 158 ± 6 nM). Mass spectrometry-based kinome profiling shows PROTAC 2 selectively degrades CDK9 in MiaPaCa2 cells and sensitizes them to Venetoclax mediated growth inhibition.

deficiency leads to early embryonic lethality in mice and defects in cell cycle progression in MEFs.

RNA polymerase II subunit A Carboxy-Terminal Domain Phosphatase 1 (CTDP1), a member of the haloacid dehalogenase superfamily phosphatases, has a defined role in transcriptional regulation, but emerging evidence suggests an expanded functional repertoire in the cell cycle and DNA damage response. In humans, a splice site mutation in gives rise to the rare Congenital Cataracts Facial Dysmorphism and Neuropathy syndrome, and recent evidence from our lab indicates CTDP1 is required for breast cancer growth and proliferation. To explore the physiological function of CTDP1 in a mammalian system, we generated a conditional knockout mouse model by insertion of sites upstream of exon 3 and downstream of exon 4.

Determination of Oxyphylla A Enantiomers in the Fruits of by a Chiral High-Performance Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Method and Comparison of Their In Vivo Biological Activities.

()-Oxyphylla A, a natural product isolated from Miquel as a food and medicinal plant, has been reported previously as a novel chiral compound that possesses a potential therapeutic value for Parkinson's disease (PD). A chiral high-performance liquid chromatography-multiple reaction monitoring-mass spectrometry method was developed to separate oxyphylla A enantiomers and to identify the presence of natural ()-oxyphylla A for the first time. Twelve samples of dried fruits were analyzed in which a large variation in the abundance of enantiomers was observed.

Implementation of a multiple-fraction concatenation strategy in an online two-dimensional high-/low-pH reversed-phase/reversed-phase liquid chromatography platform for qualitative and quantitative shotgun proteomic analyses.

Multidimensional liquid chromatography is the mainstay separation technique used for shotgun proteomic analyses. The application of a multiple-fraction concatenation (MFC) strategy can result in a more disperse and consistent peptide elution profile across different fractions, when compared with a conventional strategy. Herein, we present the first automated online RP-RP platform implementing an MFC strategy to facilitate robust, unattended, routine proteomic analyses.

UBR5 HECT domain mutations identified in mantle cell lymphoma control maturation of B cells.

Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next-generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including mutations in the ubiquitin E3 ligase UBR5.

The Proteomic Landscape of Pancreatic Ductal Adenocarcinoma Liver Metastases Identifies Molecular Subtypes and Associations with Clinical Response.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease that can be separated into distinct subtypes based on molecular signatures. Identifying PDAC subtype-specific therapeutic vulnerabilities is necessary to develop precision medicine approaches to treat PDAC.

Experimental Design: A total of 56 PDAC liver metastases were obtained from the UNMC Rapid Autopsy Program and analyzed with quantitative proteomics.

Delineating the role of FANCA in glucose-stimulated insulin secretion in β cells through its protein interactome.

Hyperinsulinemia affects 72% of Fanconi anemia (FA) patients and an additional 25% experience lowered glucose tolerance or frank diabetes. The underlying molecular mechanisms contributing to the dysfunction of FA pancreas β cells is unknown. Therefore, we sought to evaluate the functional role of FANCA, the most commonly mutated gene in FA, in glucose-stimulated insulin secretion (GSIS).

The Selection of a Hepatocyte Cell Line Susceptible to Sporozoite Invasion That Is Associated With Expression of Glypican-3.

studies of liver stage (LS) development of the human malaria parasite are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to sporozoite invasion, we created an optimized system by mimicking liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3-5% and early development of exoerythrocytic forms-a 3- to 5-fold improvement on current hepatocarcinoma models for invasion.

Paraquat-Mediated Oxidative Stress in Mosquitoes Is Regulated by An Endoplasmic Reticulum (ER) Stress Response.

Paraquat is a potent superoxide (O₂)-inducing agent that is capable of inducing an oxidative imbalance in the mosquito midgut. This oxidative imbalance can super-stress the malaria parasite, leading to arrested development in the mosquito midgut and reduced transmission. While several studies have explored the effect of paraquat on malaria parasites, a fundamental understanding of the mosquito response to this compound remains unknown.

A diagnostic autoantibody signature for primary cutaneous melanoma.

Melanoma is an aggressive form of skin cancer that is curable by surgical excision in the majority of cases, if detected at an early stage. To improve early stage melanoma detection, the development of a highly sensitive diagnostic test is of utmost importance. Here we aimed to identify antibodies to a panel of tumour associated antigens that can differentiate primary melanoma patients and healthy individuals.

Tetramethylpyrazine nitrone, a multifunctional neuroprotective agent for ischemic stroke therapy.

TBN, a novel tetramethylpyrazine derivative armed with a powerful free radical-scavenging nitrone moiety, has been reported to reduce cerebral infarction in rats through multi-functional mechanisms of action. Here we study the therapeutic effects of TBN on non-human primate model of stroke. Thirty male Cynomolgus macaques were subjected to stroke with 4 hours ischemia and then reperfusion.

Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-glycomics analysis.

In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-glycomics analyses. The performance of the 3D system alone was benchmarked through the analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets.

Fully automated multidimensional reversed-phase liquid chromatography with tandem anion/cation exchange columns for simultaneous global endogenous tyrosine nitration detection, integral membrane protein characterization, and quantitative proteomics mapping in cerebral infarcts.

Protein tyrosine nitration (PTN) is a signature hallmark of radical-induced nitrative stress in a wide range of pathophysiological conditions, with naturally occurring abundances at substoichiometric levels. In this present study, a fully automated four-dimensional platform, consisting of high-/low-pH reversed-phase dimensions with two additional complementary, strong anion (SAX) and cation exchange (SCX), chromatographic separation stages inserted in tandem, was implemented for the simultaneous mapping of endogenous nitrated tyrosine-containing peptides within the global proteomic context of a Macaca fascicularis cerebral ischemic stroke model. This integrated RP-SA(C)X-RP platform was initially benchmarked through proteomic analyses of Saccharomyces cerevisiae, revealing extended proteome and protein coverage.

A versatile reversed phase-strong cation exchange-reversed phase (RP-SCX-RP) multidimensional liquid chromatography platform for qualitative and quantitative shotgun proteomics.

An automatable, robust, high-performance online multidimensional liquid chromatography (MDLC) platform comprising of pH 10 reversed-phase (RP), strong cation exchange (SCX), and pH 2 RP separation stages has been integrated into a modified commercial off-the-shelf LC instrument with a simple rewiring, enabling accelerated routine qualitative and quantitative proteomics analyses. This system has been redesigned with a dual-trap column configuration to improve the throughput by greatly decreasing the system idle time. The performance of this new design has been benchmarked through analysis of the total lysate of S.

Goldfish spexin: solution structure and novel function as a satiety factor in feeding control.

Spexin (SPX) is a neuropeptide identified recently by bioinformatic approach. At present not much is known about its biological actions, and comparative studies of SPX in nonmammalian species are still lacking. To examine the structure and function of SPX in fish model, SPX was cloned in goldfish and found to be highly comparable with its mammalian counterparts.