Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down mass spectrometry (MS). Improved sequence coverage, critical for reliable annotation of posttranslational modifications and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events.

Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS.

Negative-Ion Electron Capture Dissociation of MALDI-Generated Peptide Anions.

Negative-ion electron capture dissociation (niECD) is an anion MS/MS technique that provides fragmentation analogous to conventional ECD, including high peptide sequence coverage and retention of labile post-translational modifications (PTMs). niECD has been proposed to be the most efficient for salt-bridged zwitterionic precursor ion structures. Several important PTMs, e.

Cancer cell survival depends on collagen uptake into tumor-associated stroma.

Collagen I, the most abundant protein in humans, is ubiquitous in solid tumors where it provides a rich source of exploitable metabolic fuel for cancer cells. While tumor cells were unable to exploit collagen directly, here we show they can usurp metabolic byproducts of collagen-consuming tumor-associated stroma. Using genetically engineered mouse models, we discovered that solid tumor growth depends upon collagen binding and uptake mediated by the TEM8/ANTXR1 cell surface protein in tumor-associated stroma.

MALDI imaging directed laser ablation tissue microsampling for data independent acquisition proteomics.

A multimodal workflow for mass spectrometry imaging was developed that combines MALDI imaging with protein identification and quantification by liquid chromatography tandem mass spectrometry (LC-MS/MS). Thin tissue sections were analyzed by MALDI imaging, and the regions of interest (ROI) were identified using a smoothing and edge detection procedure. A midinfrared laser at 3-μm wavelength was used to remove the ROI from the brain tissue section after MALDI mass spectrometry imaging (MALDI MSI).

Piezoelectric matrix-assisted ionization.

We have developed a new actuation method for matrix-assisted ionization with good temporal and spatial resolution using piezoelectric cantilever. A strike from the piezoelectric bimorph cantilever on a thin metal foil was used to remove materials deposited on the opposite side facing the mass spectrometer inlet. Highly charged ions of peptides and proteins were generated from dried droplet deposits and sampled into the inlet of the mass spectrometer.

Tumor stroma-targeted antibody-drug conjugate triggers localized anticancer drug release.

Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site.

Whole body vibration on people with sequelae of polio.

Purpose: The purpose was to explore the feasibility of whole body vibration (WBV) on polio survivors with/without post-polio syndrome (PPS) by studying its effects on walking speed (10-m walk test), endurance (2-min walk test), pain severity/interference (Brief Pain Inventory [BPI]), sleep quality (Pittsburg Sleep Quality Index), fatigue (Fatigue Severity Scale), leg strength (manual muscle testing and hand-held dynamometry), and muscle cramping (written logs).

Methods: Fifteen individuals completed the study, participating in eight sessions in two 4-week blocks. Participants started with ten 1-min vibration bouts/session, increasing to 20 min.

TEM8/ANTXR1-Specific CAR T Cells as a Targeted Therapy for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive disease lacking targeted therapy. In this study, we developed a CAR T cell-based immunotherapeutic strategy to target TEM8, a marker initially defined on endothelial cells in colon tumors that was discovered recently to be upregulated in TNBC. CAR T cells were developed that upon specific recognition of TEM8 secreted immunostimulatory cytokines and killed tumor endothelial cells as well as TEM8-positive TNBC cells.

Eradication of Tumors through Simultaneous Ablation of CD276/B7-H3-Positive Tumor Cells and Tumor Vasculature.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature.

Anthrax Toxin Receptor 1 Is Essential for Arteriogenesis in a Mouse Model of Hindlimb Ischemia.

Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1(-/-) mice.

Bursts of Bipolar Microsecond Pulses Inhibit Tumor Growth.

Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions.

Immuno-PET imaging of tumor endothelial marker 8 (TEM8).

Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring.

Three-dimensional microfluidic collagen hydrogels for investigating flow-mediated tumor-endothelial signaling and vascular organization.

Hyperpermeable tumor vessels are responsible for elevated interstitial fluid pressure and altered flow patterns within the tumor microenvironment. These aberrant hydrodynamic stresses may enhance tumor development by stimulating the angiogenic activity of endothelial cells lining the tumor vasculature. However, it is currently not known to what extent shear forces affect endothelial organization or paracrine signaling during tumor angiogenesis.

In vitro angiogenesis induced by tumor-endothelial cell co-culture in bilayered, collagen I hydrogel bioengineered tumors.

Although successful remission has been achieved when cancer is diagnosed and treated during its earliest stages of development, a tumor that has established neovascularization poses a significantly greater risk of mortality. The inability to recapitulate the complexities of a maturing in vivo tumor microenvironment in an in vitro setting has frustrated attempts to identify and test anti-angiogenesis therapies that are effective at permanently halting cancer progression. We have established an in vitro tumor angiogenesis model driven solely by paracrine signaling between MDA-MB-231 breast cancer cells and telomerase-immortalized human microvascular endothelial (TIME) cells co-cultured in a spatially relevant manner.

A three-dimensional in vitro tumor platform for modeling therapeutic irreversible electroporation.

Irreversible electroporation (IRE) is emerging as a powerful tool for tumor ablation that utilizes pulsed electric fields to destabilize the plasma membrane of cancer cells past the point of recovery. The ablated region is dictated primarily by the electric field distribution in the tissue, which forms the basis of current treatment planning algorithms. To generate data for refinement of these algorithms, there is a need to develop a physiologically accurate and reproducible platform on which to study IRE in vitro.

Takotsubo cardiomyopathy after midodrine therapy.

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Cross-talk between endothelial and breast cancer cells regulates reciprocal expression of angiogenic factors in vitro.

Reciprocal growth factor exchange between endothelial and malignant cells within the tumor microenvironment may directly stimulate neovascularization; however, the role of host vasculature in regulating tumor cell activity is not well understood. While previous studies have examined the angiogenic response of endothelial cells to tumor-secreted factors, few have explored tumor response to endothelial cells. Using an in vitro co-culture system, we investigated the influence of endothelial cells on the angiogenic phenotype of breast cancer cells.

3D in vitro bioengineered tumors based on collagen I hydrogels.

Cells cultured within a three-dimensional (3D) in vitro environment have the ability to acquire phenotypes and respond to stimuli analogous to in vivo biological systems. This approach has been utilized in tissue engineering and can also be applied to the development of a physiologically relevant in vitro tumor model. In this study, collagen I hydrogels cultured with MDA-MB-231 human breast cancer cells were bioengineered as a platform for in vitro solid tumor development.

Photothermal response of human and murine cancer cells to multiwalled carbon nanotubes after laser irradiation.

This study demonstrates the capability of multiwalled carbon nanotubes (MWNTs) coupled with laser irradiation to enhance treatment of cancer cells through enhanced and more controlled thermal deposition, increased tumor injury, and diminished heat shock protein (HSP) expression. We also explored the potential promise of MWNTs as drug delivery agents by observing the degree of intracellular uptake of these nanoparticles. To determine the heat generation capability of MWNTs, the absorption spectra and temperature rise during heating were measured.

Long-term survival following a single treatment of kidney tumors with multiwalled carbon nanotubes and near-infrared radiation.

Multiwalled carbon nanotubes (MWCNTs) exhibit physical properties that render them ideal candidates for application as noninvasive mediators of photothermal cancer ablation. Here, we demonstrate that use of MWCNTs to generate heat in response to near-infrared radiation (NIR) results in thermal destruction of kidney cancer in vitro and in vivo. We document the thermal effects of the therapy through magnetic resonance temperature-mapping and heat shock protein-reactive immunohistochemistry.