Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification.

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC-MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC-MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot's Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

Rewiring of cortical glucose metabolism fuels human brain cancer growth.

The brain avidly consumes glucose to fuel neurophysiology. Cancers of the brain, such as glioblastoma (GBM), lose aspects of normal biology and gain the ability to proliferate and invade healthy tissue. How brain cancers rewire glucose utilization to fuel these processes is poorly understood. Here we perform infusions of 13 C-labeled glucose into patients and mice with brain cancer to define the metabolic fates of glucose-derived carbon in tumor and cortex. By combining these measurements with quantitative metabolic flux analysis, we find that human cortex funnels glucose-derived carbons towards physiologic processes including TCA cycle oxidation and neurotransmitter synthesis. In contrast, brain cancers downregulate these physiologic processes, scavenge alternative carbon sources from the environment, and instead use glucose-derived carbons to produce molecules needed for proliferation and invasion. Targeting this metabolic rewiring in mice through dietary modulation selectively alters GBM metabolism and slows tumor growth. Significance: This study is the first to directly measure biosynthetic flux in both glioma and cortical tissue in human brain cancer patients. Brain tumors rewire glucose carbon utilization away from oxidation and neurotransmitter production towards biosynthesis to fuel growth. Blocking these metabolic adaptations with dietary interventions slows brain cancer growth with minimal effects on cortical metabolism.

Structural elucidation and isomeric differentiation/quantitation of monophosphorylated phosphoinositides using gas-phase ion/ion reactions and dissociation kinetics.

Phosphoinositides, phosphorylated derivatives of phosphatidylinositols, are essential signaling phospholipids in all mammalian cellular membranes. With three known phosphorylated derivatives of phosphatidylinositols at the 3-, 4-, and 5-positions along the myo-inositol ring, various fatty acyl chain lengths, and varying degrees of unsaturation, numerous isomers can be present. It is challenging for shotgun-MS to accurately identify and characterize phosphoinositides and their isomers using the most readily available precursor ion types. To overcome this challenge, novel gas-phase ion/ion chemistry was used to expand the range of precursor ion-types for subsequent structural characterization of phosphoinositides using shot-gun tandem mass spectrometry. The degree of phosphorylation and fatty acyl sum composition are readily obtained by ion-trap CID of deprotonated phosphoinositides. Carbon-carbon double bond position of the fatty acyl chains can be localized via a charge inversion ion/ion reaction. Utilizing sequential ion/ion reactions and subsequent activation yields product ion information that is of limited utility for phosphorylation site localization. However, the kinetics of dissociation allowed for isomeric differentiation of the position of the phosphate group. Furthermore, employing the same kinetics method, relative quantitative information was gained for the isomeric species.

Conformer-Specific Spectroscopy and IR-Induced Isomerization of a Model γ-Peptide: Ac-γ4-Phe-NHMe.

Single-conformation IR and UV spectroscopy of the prototypical capped γ-peptide Ac-γ4-Phe-NHMe (γ4F) was carried out under jet-cooled conditions in the gas phase in order to understand its innate conformational preferences in the absence of a solvent. We obtained conformer-specific IR and UV spectra and compared the results with calculations to make assignments and explore the differences between the γ2- and γ4-substituted molecules. We found four conformers of γ4F in our experiment. Three conformers form nine-membered hydrogen-bonded rings (C9) enclosed by an NH···O═C H-bond but differing in their phenyl ring positions (a, g+, and g-). The fourth conformer forms a strained seven-membered hydrogen-bonded ring in which the amide groups lie in a nominally anti-parallel arrangement stacked on top of one another (labeled S7). This conformer is a close analogue of the amide-stacked conformer (S) found previously in γ2F, in which the Phe side chain is substituted at the γ2 position, Ac-γ2-Phe-NHMe (J. Am. Chem. Soc. 2009, 131, 14243-14245). IR population transfer spectroscopy was used to determine the fractional abundances of the γ4F conformers in the expansion. A combination of force field and density functional theory calculations is used to map out the conformational potential energy surfaces for γ4F and compare it with its γ2F counterpart. Based on this analysis, the phenyl ring prefers to take up structures that facilitate NH···π interactions in γ4F or avoid phenyl interactions with the C═O group in γ2F. The disconnectivity graph for γ4F reveals separate basins associated with the C9 and amide-stacked conformational families, which are separated by a barrier of about 42 kJ/mol. The overall shape of the potential energy surface bears a resemblance to peptides and proteins that have a misfolding pathway that competes with the formation of the native structure.

Gas-Phase Covalent Bond Formation via Nucleophilic Substitution: A Dissociation Kinetics Study of Leaving Groups, Isomeric R Groups, and Nucleophilic Sites.

Nucleophilic substitution covalent modification ion/ion reactions were carried out in a linear quadrupole ion trap between the doubly protonated peptides KGAILKGAILR, RARARAA, and RKRARAA and isomers of either singly deprotonated 3- or 4-sulfobenzoic acid (n-SBA) esterified with either N-hydroxysuccinimide (NHS) or 1-hydroxy-7-aza-benzotriazole (HOBt). The cation/anion attachment product, through which the covalent reaction occurs, was isolated and subjected to dipolar DC (DDC) activation to generate covalently modified product over the ranges of DDC activation energies and times. The resulting survival yields were used to determine reaction rates, and Tolmachev's effective ion temperature was used to extract Arrhenius and Eyring activation parameters. It was found that the kinetics determined under these conditions are highly sensitive to the identities and locations of the nucleophilic sites on the peptides, the leaving groups on the reagent, and the location of the attachment sites on the reagent and analyte. Depending upon the identity of the analyte/reagent combination, significant variations in activation energy or entropy (or both) were both found to underlie the measured rate differences. The determination of dissociation kinetics under DDC conditions and application of Tolmachev's effective ion temperature treatment enables unique insights into the dynamics of gas-phase covalent bond formation via ion/ion reactions.

Resolving Isomers of Star-Branched Poly(Ethylene Glycols) by IMS-MS Using Multiply Charged Ions.

Ion mobility spectrometry (IMS) mass spectrometry (MS) centers on the ability to separate gaseous structures by size, charge, shape, and followed by mass-to-charge (m/z). For oligomeric structures, improved separation is hypothesized to be related to the ability to extend structures through repulsive forces between cations electrostatically bonded to the oligomers. Here we show the ability to separate differently branched multiply charged ions of star-branched poly(ethylene glycol) oligomers (up to 2000 Da) regardless of whether formed by electrospray ionization (ESI) charged solution droplets or from charged solid particles produced directly from a surface by matrix-assisted ionization. Detailed structural characterization of isomers of the star-branched compositions was first established using a home-built high-resolution ESI IMS-MS instrument. The doubly charged ions have well-resolved drift times, achieving separation of isomers and also allowing differentiation of star-branched versus linear oligomers. An IMS-MS "snapshot" approach allows visualization of architectural dispersity and (im)purity of samples in a straightforward manner. Analyses capabilities are shown for different cations and ionization methods using commercially available traveling wave IMS-MS instruments. Analyses directly from surfaces using the new ionization processes are, because of the multiply charging, not only associated with the benefits of improved gas-phase separations, relative to that of ions produced by matrix-assisted laser desorption/ionization, but also provide the potential for spatially resolved measurements relative to ESI and other ionization methods.

Optimization of histone deacetylase inhibitor activity in non-secosteroidal vitamin D-receptor agonist hybrids.

The combination of (1α,25)-dihydroxyvitamin D3 (1,25D) and histone deacetylase inhibitor (HDACi) trichostatin A is highly antiproliferative in numerous cancer cell lines. We have previously prepared novel non-secosteroidal hybrid molecules which simultaneously act as both vitamin D receptor (VDR) agonists and HDACi. These molecules function as cytostatic and cytotoxic agents in 1,25D-resistant SCC4 squamous carcinoma cells. Here we have extended the scope of the hybrids by making several modifications to the diarylpentane core and to the aliphatic spacer unit to develop molecules with increased potency towards HDACs while maintaining VDR agonist activity. Notably, hybrid DK-366 (33a), a direct analog of first-generation hybrids but lacking a methyl group on one aryl ring possesses low micromolar potency for HDAC3 and HDAC6 and is a highly effective antiproliferative agent in SCC4 cells. Chain extended hybrids such as DK-367 (33c) possess even greater HDAC potency and are also highly antiproliferative. These results show that we can optimize HDACi potency in hybrid molecules without sacrificing VDR agonism.

Synthetically accessible non-secosteroidal hybrid molecules combining vitamin D receptor agonism and histone deacetylase inhibition.

1,25-Dihydroxyvitamin D(3) (1,25D), the hormonal form of vitamin D, and several analogs have failed as monotherapies for cancer because of poor efficacy or acquired resistance. However, 1,25D analogs are amenable to bifunctionalization. Preclinical studies have revealed combinatorial effects of 1,25D analogs and histone deacetylase inhibitors (HDACi). Secosteroidal hybrid molecules combining vitamin D receptor (VDR) agonism with HDACi displayed enhanced efficacy but are laborious to synthesize. Here, we have developed easily assembled, fully integrated, non-secosteroidal VDR agonist/HDACi hybrids. The most promising are full VDR agonists with ~10-fold lower potency than 1,25D. Structure/function studies revealed that antiproliferative activity against 1,25D-resistant squamous carcinoma cells required VDR agonism and HDACi. Remarkably, modeling and X-ray crystallography reveal non-secosteroidal hybrids bind in the VDR ligand binding domain in the opposite orientation of their secosteroidal counterparts.

The Xpc gene markedly affects cell survival in mouse bone marrow.

The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc-/- mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc-/- and strain-matched wild-type mice. Using several independent methods, Xpc-/- bone marrow was approximately 10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc-/- mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.

Chemotherapeutic selectivity conferred by selenium: a role for p53-dependent DNA repair.

Selenium in various chemical forms has been the subject of cancer chemoprevention trials, but, more recently, selenium has been used in combination with DNA-damaging chemotherapeutics. Specifically, selenium protected tissues from dose-limiting toxicity and, in fact, allowed delivery of higher chemotherapeutic doses. At the same time, selenium did not protect cancer cells. Therefore, we seek to define the genetic basis for the observed selectivity of selenium in combination chemotherapeutics. The tumor suppressor p53 is mutated in the vast majority of cancers, but is by definition wild-type in nontarget tissues such as bone marrow and gut epithelium, tissues that are often dose-limiting due to DNA damage. We used primary, low-passage mouse embryonic fibroblasts that are wild-type or null for p53 genes to test differential effects of selenium. Seleno-l-methionine, nontoxic by itself, was used to pretreat cell cultures before exposure to UV radiation or UV-mimetic cancer chemotherapy drugs. Seleno-l-methionine pretreatment caused a DNA repair response, which protected from subsequent challenge with DNA-damaging agents. The observed DNA repair response and subsequent DNA damage protection were p53 dependent as neither was observed in p53-null cells. The data suggest that (a) p53 may be an important genetic determinant that distinguishes normal cells from cancer cells, and (b) combinatorial chemotherapeutics that act by p53-dependent mechanisms may enhance chemotherapeutic efficacy by increasing the chemotherapeutic window distinguishing cancer cells from normal cells.

Selenium protection from DNA damage involves a Ref1/p53/Brca1 protein complex.

Selenium, in the form of seleno-L-methionine (SeMet), induced Redox-factor-1 (Ref1) and p53 proteins in normal human and mouse fibroblasts. Ref1 and p53 are known to be associated with each other, resulting in enhanced sequence-specific DNA binding by p53 and transactivation of p53-regulated effector genes. SeMet preferentially induced the DNA repair branch of the p53 pathway, while apoptosis and cell cycle arrest were unaffected. Accordingly, pretreatment with SeMet protected normal fibroblasts from subsequent DNA damage. In the current study, Brca1 and Ref1 were shown to interact concurrently with p53 in targeting a SeMet-induced DNA repair response. Moreover, like p53 and Ref1, Brca1 was required for SeMet-mediated DNA damage protection, as brca1 -/- mouse fibroblasts were not protected from UV-radiation by SeMet treatment. These findings indicate that besides p53 and Ref1, Brca1 is required for selenium protection from DNA damage. The data are consistent with selective induction of the DNA repair branch of the p53 pathway by SeMet.

Fibronectin binding protein BBK32 of the Lyme disease spirochete promotes bacterial attachment to glycosaminoglycans.

Borrelia burgdorferi, the agent of Lyme disease, causes a multisystemic illness that can affect the skin, heart, joints, and nervous system and is capable of attachment to diverse cell types. Among the host components recognized by this spirochete are fibronectin and glycosaminoglycans (GAGs). Three surface-localized GAG-binding bacterial ligands, Bgp, DbpA, and DbpB, have been previously identified, but recent studies suggested that at least one additional GAG-binding ligand is expressed on the spirochetal surface when the spirochete is adapted to the mammalian host environment. BBK32 is a surface lipoprotein that is produced during infection and that has been shown to bind to fibronectin. In this study, we show that, when BBK32 was produced from a shuttle vector in an otherwise nonadherent high-passage B. burgdorferi strain, the protein localized on the bacterial surface and conferred attachment to fibronectin and to mammalian cell monolayers. In addition, the high-passage strain producing BBK32 bound to purified preparations of the GAGs dermatan sulfate and heparin, as well as to these GAGs on the surfaces of cultured mammalian cells. Recombinant BBK32 recognized purified heparin, indicating that the bacterial attachment to GAGs was due to direct binding by BBK32. This GAG-binding activity of BBK32 is apparently independent of fibronectin recognition, because exogenous heparin had no effect on BBK32-mediated bacterial binding to fibronectin.

Neointimal formation in two apolipoprotein E-deficient mouse strains with different atherosclerosis susceptibility.

C57BL/6 (B6) and C3H/HeJ (C3H) are two commonly used mouse strains that differ markedly in atherosclerosis susceptibility. In this study, we determined plaque formation after removal of the endothelium in the two strains carrying the mutant apolipoprotein E gene (apoE(-/-)). At 10 weeks of age, male B6.apoE(-/-) and C3H.apoE(-/-) mice underwent endothelial denudation of the left common carotid artery. Two weeks after injury, B6.apoE(-/-) mice developed significantly larger neointimal lesions in the vessel than their C3H.apoE(-/-) counterparts, although they had comparable plasma cholesterol levels on a chow diet. Feeding of a Western diet aggravated lesion formation in both strains, but the increase was more dramatic in B6.apoE(-/-) mice than in C3H.apoE(-/-) mice. Immunohistochemical and histological analyses demonstrated the presence of macrophage foam cells in neointimal lesions. We then compared neointimal growth in F1 mice reconstituted with bone marrow from B6.apoE(-/-) and C3H.apoE(-/-) mice. No significant lesions were observed 2 weeks after endothelial denudation in the mice reconstituted with bone marrow from either donor. Thus, these data indicate that foam cell formation contributes to neointimal growth in the hyperlipidemic apoE(-/-) model and that neither endothelial cells nor blood cells alone explain the dramatic difference between B6 and C3H mice in plaque formation.

Radical carboxyarylation approach to lignans. Total synthesis of (-)-arctigenin, (-)-matairesinol, and related natural products.

[reaction: see text] Total syntheses of seven biologically important lignan natural products, including (-)-arctigenin, (-)-matairesinol, and (-)-alpha-conidendrin, by way of a highly stereoselective domino radical sequence is presented. The reported stereochemistry of the natural product 7-hydroxyarctigenin is shown to be erroneous; a diastereoisomeric structure is assigned to the natural product.

Decorin-binding proteins A and B confer distinct mammalian cell type-specific attachment by Borrelia burgdorferi, the Lyme disease spirochete.

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. In the current study, we show that the expression of decorin-binding protein (Dbp) A and/or DbpB, two B. burgdorferi surface proteins that bind GAGs, is sufficient to convert a high-passage nonadherent B. burgdorferi strain into one that efficiently binds 293 epithelial cells. Epithelial cell attachment was mediated by dermatan sulfate, and, consistent with this GAG-binding specificity, these recombinant strains did not bind EA-Hy926 endothelial cells. The GAG-binding properties of bacteria expressing DbpB or DbpA were distinguishable, and DbpB but not DbpA promoted spirochetal attachment to C6 glial cells. Thus, DbpA and DbpB may each play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains may provide a relatively simple genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.