Glucose-induced CRL4COP1-p53 axis amplifies glycometabolism to drive tumorigenesis.

The diabetes-cancer association remains underexplained. Here, we describe a glucose-signaling axis that reinforces glucose uptake and glycolysis to consolidate the Warburg effect and overcome tumor suppression. Specifically, glucose-dependent CK2 O-GlcNAcylation impedes its phosphorylation of CSN2, a modification required for the deneddylase CSN to sequester Cullin RING ligase 4 (CRL4). Glucose, therefore, elicits CSN-CRL4 dissociation to assemble the CRL4COP1 E3 ligase, which targets p53 to derepress glycolytic enzymes. A genetic or pharmacologic disruption of the O-GlcNAc-CK2-CSN2-CRL4COP1 axis abrogates glucose-induced p53 degradation and cancer cell proliferation. Diet-induced overnutrition upregulates the CRL4COP1-p53 axis to promote PyMT-induced mammary tumorigenesis in wild type but not in mammary-gland-specific p53 knockout mice. These effects of overnutrition are reversed by P28, an investigational peptide inhibitor of COP1-p53 interaction. Thus, glycometabolism self-amplifies via a glucose-induced post-translational modification cascade culminating in CRL4COP1-mediated p53 degradation. Such mutation-independent p53 checkpoint bypass may represent the carcinogenic origin and targetable vulnerability of hyperglycemia-driven cancer.

Viscoelastic-Sorting Integrated Deformability Cytometer for High-Throughput Sorting and High-Precision Mechanical Phenotyping of Tumor Cells.

The counts and phenotypes of circulating tumor cells (CTCs) in whole blood are useful for disease monitoring and prognostic assessment of cancer. However, phenotyping CTCs in the blood is difficult due to the presence of a large number of background blood cells, especially some blood cells with features similar to those of tumor cells. Herein, we presented a viscoelastic-sorting integrated deformability cytometer (VSDC) for high-throughput label-free sorting and high-precision mechanical phenotyping of tumor cells. A sorting chip for removing large background blood cells and a detection chip for detecting multiple cellular mechanical properties were integrated into our VSDC. Our VSDC has a sorting efficiency and a purity of over 95% and over 81% for tumor cells, respectively. Furthermore, multiple mechanical parameters were used to distinguish tumor cells from white blood cells using machine learning. An accuracy of over 97% for identifying tumor cells was successfully achieved with the highest identification accuracy of 99.4% for MCF-7 cells. It is envisioned that our VSDC will open up new avenues for high-throughput and label-free single-cell analysis in various biomedical applications.

Simultaneous Mapping of Amino Neurotransmitters and Nucleoside Neuromodulators on Brain Tissue Sections by On-Tissue Chemoselective Derivatization and MALDI-MSI.

Neurotransmitters (NTs) and neuromodulators (NMs) are two of the most important neurochemicals in the brain, and their imbalances in specific brain regions are thought to underlie certain neurological disorders. We present an on-tissue chemoselective derivatization mass spectrometry imaging (OTCD-MSI) method for the simultaneous mapping of NTs and NMs. Our derivatization system consists of a pyridiniumyl-benzylboronic acid based derivatization reagent and pyrylium salt, which facilitate covalent charge labeling of molecules containing cis-diol and primary amino, respectively. These derivatization systems improved the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MSI and simplified the identification of amino NTs and nucleoside NMs by the innate chemoselectivity of derivatization reagents and the unique isotopic pattern of boron-derivative reagents. We demonstrated the ability of the developed method on brain sections from a hypoxia mouse model and control. The simultaneous imaging of NTs and NMs provided a method for exploring how hypoxic stress and drugs affect specific brain regions through neurotransmitter modulation.

RHOJ as a novel mechanosensitive modulator of endothelial inflammation.

Physiological high shear stress (HSS), a frictional force generated by flowing blood, is essential for endothelial homeostasis under normal physiological conditions. HSS suppresses atherosclerosis by inhibiting endothelial inflammation. However, the molecular mechanisms underlying this process have not been fully elucidated. Here, we report that HSS downregulated the mRNA and protein levels of ras homolog family member J (RHOJ) in endothelial cells (ECs). Silencing endogenous RHOJ expression decreased the mRNA and protein levels of proinflammatory vascular cell adhesion molecule 1 (VCAM-1) and intercellular cell adhesion molecule 1 (ICAM-1) in ECs, leading to a reduction in monocyte adhesion to ECs. Conversely, the overexpression of RHOJ had the opposite effect. RNA-sequencing analysis uncovered several differentially expressed genes (such as yes-associated protein 1 (YAP1),heme oxygenase-1 (HO1), and monocyte chemoattractant protein-1 (MCP1)) and pathways (such as nuclear factor-kappa B (NF-κB), fluid shear stress and atherosclerosis, and cell adhesion pathways) as RHOJ targets. Additionally, HSS was observed to alleviate endothelial inflammation by inhibiting RHOJ expression. Finally, methylated RNA immunoprecipitation sequencing (MeRIP-seq) illustrated that fluid shear stress regulates RHOJ expression in an N6-methyladenosine (m6A)-dependent manner. Mechanistically, the RNA m6A writer, methyltransferase 3 (METTL3), and the RNA m6A readers, YTH N6-methyladenosine RNA-binding protein F 3 (YTHDF3) and YTH N6-methyladenosine RNA-binding protein C 1/2 (YTHDC1/2), are involved in this process. Taken together, our data demonstrate that HSS-induced downregulation of RHOJ contributes to endothelial homeostasis by suppressing endothelial inflammation and that RHOJ inhibition in ECs is a promising therapeutic strategy for endothelial dysfunction.

N6-Methyladenosine Demethylase FTO (Fat Mass and Obesity-Associated Protein) as a Novel Mediator of Statin Effects in Human Endothelial Cells.

BACKGROUND: N6-methyladenosine (m6A) plays a critical role in various biological processes. However, no study has addressed the role of m6A modification in the statin-induced protection of endothelial cells (ECs). METHODS: Quantitative real-time polymerase chain reaction and Western blotting analyses were used to study the expression of m6A regulatory genes in atorvastatin-treated ECs. Gain- and loss-of-function assays, methylated RNA immunoprecipitation analysis, and dual-luciferase reporter assays were performed to clarify the function of FTO (fat mass and obesity-associated protein) in ECs. RESULTS: Atorvastatin decreased FTO protein expression in ECs. The knockdown of FTO enhanced the mRNA and protein expression of KLF2 (Kruppel-like factor 2) and eNOS (endothelial NO synthase) but attenuated TNFα (tumor necrosis factor alpha)-induced VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) expression, as well as the adhesion of monocytes to ECs. Conversely, FTO overexpression significantly upregulated the mRNA and protein levels of VCAM-1 and ICAM-1, downregulated those of KLF2 and eNOS, and strongly attenuated the atorvastatin-mediated induction of KLF2 and eNOS expression. Subsequent investigations demonstrated that KLF2 and eNOS are functionally critical targets of FTO. Mechanistically, FTO interacted with KLF2 and eNOS transcripts and regulated their expression in an m6A-dependent manner. After FTO silencing, KLF2 and eNOS transcripts with higher levels of m6A modification in their 3' untranslated regions were captured by YTHDF3 (YT521-B homology m6A RNA-binding protein 3), resulting in mRNA stabilization and the induction of KLF2 and eNOS protein expression. CONCLUSIONS: FTO might serve as a novel molecular target to modulate endothelial function in vascular diseases.

Theory of all-coupling angulon for molecules rotating in many-body environment.

The formation of angulon, stemming from the rotor (molecule or impurity), rotating in the quantum many-body field, adds a new member to the quasi-particles' family and has aroused intense interest in multiple research fields. However, the analysis of the coupling strength between the rotor and its hosting environment remains a challenging task, both in theory and experiment. Here, we develop the all-coupling theory of the angulon by introducing a unitary transformation, where the renormalization of the rotational constants for different molecules in the helium nanodroplets is reproduced, getting excellent agreement with the experimental data collected during the past decades. Moreover, the strength of molecule-helium coupling and the effective radius of the solvation shell co-rotating along with the molecular rotor could be estimated qualitatively. This model not only provides significant enlightenment for analyzing the rotational spectroscopy of molecules in the phononic environment, but also provides a new method to study the transfer of the phonon angular momentum in the angulon frame.

Basis for metabolite-dependent Cullin-RING ligase deneddylation by the COP9 signalosome.

The Cullin-RING ligases (CRLs) are the largest family of ubiquitin E3s activated by neddylation and regulated by the deneddylase COP9 signalosome (CSN). The inositol polyphosphate metabolites promote the formation of CRL-CSN complexes, but with unclear mechanism of action. Here, we provide structural and genetic evidence supporting inositol hexakisphosphate (IP6) as a general CSN cofactor recruiting CRLs. We determined the crystal structure of IP6 in complex with CSN subunit 2 (CSN2), based on which we identified the IP6-corresponding electron density in the cryoelectron microscopy map of a CRL4A-CSN complex. IP6 binds to a cognate pocket formed by conserved lysine residues from CSN2 and Rbx1/Roc1, thereby strengthening CRL-CSN interactions to dislodge the E2 CDC34/UBE2R from CRL and to promote CRL deneddylation. IP6 binding-deficient Csn2K70E/K70E knockin mice are embryonic lethal. The same mutation disabled Schizosaccharomyces pombe Csn2 from rescuing UV-hypersensitivity of csn2-null yeast. These data suggest that CRL transition from the E2-bound active state to the CSN-bound sequestered state is critically assisted by an interfacial IP6 small molecule, whose metabolism may be coupled to CRL-CSN complex dynamics.

Bimetallic Anionic Organic Frameworks with Solid-State Cation Conduction for Charge Storage Applications.

A new phosphonate-based anionic bimetallic organic framework, with the general formula of A4 -Zn-DOBDP (wherein A is Li+ or Na+ , and DOBDP6- is the 2,5-dioxido-1,4-benzenediphosphate ligand) is prepared and characterized for energy storage applications. With four alkali cations per formula unit, the A4 -Zn-DOBDP MOF is found to be the first example of non-solvated cation conducting MOF with measured conductivities of 5.4×10-8  S cm-1 and 3.4×10-8  S cm-1 for Li4 - and Na4 - phases, indicating phase and composition effects of Li+ and Na+ shuttling through the channels. Three orders of magnitude increase in ionic conductivity is further attained upon solvation with propylene carbonate, placing this system among the best MOF ionic conductors at room temperature. As positive electrode material, Li4 -Zn-DOBDP delivers a specific capacity of 140 mAh g-1 at a high average discharge potential of 3.2 V (vs. Li+ /Li) with 90 % of capacity retention over 100 cycles. The significance of this research extends from the development of a new family of electroactive phosphonate-based MOFs with inherent ionic conductivity and reversible cation storage, to providing elementary insights into the development of highly sought yet still evasive MOFs with mixed-ion and electron conduction for energy storage applications.

A Beclin 1-targeting stapled peptide synergizes with erlotinib to potently inhibit proliferation of non-small-cell lung cancer cells.

Epidermal Growth Factor Receptor (EGFR) is a major drug target for non-small-cell lung carcinoma (NSCLC). Tyrosine Kinase Inhibitors (TKIs) like erlotinib are potent inhibitors of EGFR and have achieved impressive clinical success against NSCLC. However, NSCLC cells readily develop resistance to TKIs by acquiring mutations in EGFR or other oncogenes. Novel strategies to inhibit EGFR are needed to overcome this urgent problem of TKI resistance. Beclin 1 is an essential autophagy protein and is intimately involved in tumorigenesis and EGFR signaling. Here we present data to show that a Beclin 1-targeting stapled peptide Tat-SP4 can inhibit the EGFR signaling pathway by enhancing the Beclin 1-mediated endolysosomal degradation of EGFR. This inhibition mechanism is orthogonal to that employed by TKIs and is effective against either wild-type or mutant EGFR. Tat-SP4 alone showed moderate anti-proliferative efficacy in NSCLC cells but synergized with erlotinib to potently inhibit NSCLC proliferation. These results suggest that Beclin 1-targeting stapled peptides may be used in combination with TKIs to enhance their efficacy, particularly for NSCLC subtypes refractory to current regiments.

Suramin and NF449 are IP5K inhibitors that disrupt inositol hexakisphosphate-mediated regulation of cullin-RING ligase and sensitize cancer cells to MLN4924/pevonedistat.

Inositol hexakisphosphate (IP6) is an abundant metabolite synthesized from inositol 1,3,4,5,6-pentakisphosphate (IP5) by the single IP5 2-kinase (IP5K). Genetic and biochemical studies have shown that IP6 usually functions as a structural cofactor in protein(s) mediating mRNA export, DNA repair, necroptosis, 3D genome organization, HIV infection, and cullin-RING ligase (CRL) deneddylation. However, it remains unknown whether pharmacological perturbation of cellular IP6 levels affects any of these processes. Here, we performed screening for small molecules that regulate human IP5K activity, revealing that the antiparasitic drug and polysulfonic compound suramin efficiently inhibits IP5K in vitro and in vivo The results from docking experiments and biochemical validations suggested that the suramin targets IP5K in a distinct bidentate manner by concurrently binding to the ATP- and IP5-binding pockets, thereby inhibiting both IP5 phosphorylation and ATP hydrolysis. NF449, a suramin analog with additional sulfonate moieties, more potently inhibited IP5K. Both suramin and NF449 disrupted IP6-dependent sequestration of CRL by the deneddylase COP9 signalosome, thereby affecting CRL activity cycle and component dynamics in an IP5K-dependent manner. Finally, nontoxic doses of suramin, NF449, or NF110 exacerbate the loss of cell viability elicited by the neddylation inhibitor and clinical trial drug MLN4924/pevonedistat, suggesting synergistic ef-fects. Suramin and its analogs provide structural templates for designing potent and specific IP5K inhibitors, which could be used in combination therapy along with MLN4924/pevonedistat. IP5K is a potential mechanistic target of suramin, accounting for suramin's therapeutic effects.

α-Active Pyrylium Salt 2,4,5-Triphenylpyrylium for Improved Mass Spectrometry-Based Detection of Peptides.

Pyrylium salts are considered efficient chemical tags for amino groups. However, the apparent steric selectivity of pyrylium salts limits their application in the field of chemical labeling, especially during the labeling of sterically hindered compounds like amino acids, peptides, and proteins. Herein, we have investigated the effects of the α-substitution of pyrylium salts on their reactivity. We have also investigated the mechanism of nucleophilic reactions with pyrylium salts and further proposed that the reactivity of pyrylium salts mainly depends on the position and type of their substituents. A series of pyrylium salts were synthesized, and a highly active α-monosubstituted pyrylium salt, 2,4,5-triphenylpyrylium, was developed for efficient chemical labeling. All of the 15 amino acids studied were efficiently labeled under optimized reaction conditions. The 2,4,5-triphenylpyrylium salt was highly efficient in comparison to the previously reported 2,4,6-triphenylpyrylium salt developed for lysine-specific modifications. Furthermore, we successfully used 2,4,5-triphenylpyrylium salt for the hydrophobic labeling of peptides and protein hydrolysates. The most striking observation was that the ionization efficiency of short-chain multilabeled peptides in mixed samples, after derivatization, increased by up to 60 times. The increase in ionization efficiency gradually decreased with increasing peptide chain length. During the "soft" collision-induced dissociation (CID) process, the peptide was tagged at the N-terminus with 2,4,5-triphenylpyrylium, producing abundant a-type ions and b-type ions (Δ = 28), which eases the peptide resequencing process and assists in cracking the peptide codes. Moreover, 2,4,5-triphenylpyrylium has been utilized for the proteomic analysis of HeLa cell digests. In addition, 215 additional proteins were identified in the labeled products and the coverage of most proteins was improved.

Functionally distinct IFN-γ+ IL-17A+ Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response.

Immunopathogenic roles for both Th1 (CD4+ IFN-γ+ ) and Th17 (CD4+ IL-17A+ ) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4+ T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.6 ± 4.2%) and draining LNs (mean 8.4 ± 3.9%; p = 0.01) relative to controls. Topical dexamethasone treatment effectively reduced EAU severity and decreased retinal Th1 cells (p = 0.01), but had no impact on retinal Th17/Th1 or Th17 cells compared to saline controls. Using in vitro migration assays with mouse CNS endothelium, we demonstrated that Th17/Th1 cells were significantly increased within the migrated population relative to controls (mean 15.6 ± 9.5% vs. 1.9 ± 1.5%; p = 0.01). Chemokine receptor profiles of Th17/Th1 cells (CXCR3 and CCR6) did not change throughout the transendothelial migration process and were unaffected by dexamethasone treatment. These findings support a role for Th17/Th1 cells in EAU and their resistance to steroid inhibition suggests the importance of targeting both Th17 and Th17/Th1 cells for improving therapy.

Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis.

BACKGROUND: The integrin VLA-4 (α4ß1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4ß1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. METHODS: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. RESULTS: There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. CONCLUSIONS: This α4ß1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4ß1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

Bidentatide, a Novel Plant Peptide Derived from Achyranthes bidentata Blume: Isolation, Characterization, and Neuroprotection through Inhibition of NR2B-Containing NMDA Receptors.

Increasing attention is being focused on the use of polypeptide-based N-methyl-d-aspartate (NMDA) receptor antagonists for the treatment of nervous system disorders. In our study on Achyranthes bidentata Blume, we identified an NMDA receptor subtype 2B (NR2B) antagonist that exerts distinct neuroprotective actions. This antagonist is a 33 amino acid peptide, named bidentatide, which contains three disulfide bridges that form a cysteine knot motif. We determined the neuroactive potential of bidentatide by evaluating its in vitro effects against NMDA-mediated excitotoxicity. The results showed that pretreating primary cultured hippocampal neurons with bidentatide prevented NMDA-induced cell death and apoptosis via multiple mechanisms that involved intracellular Ca2+ inhibition, NMDA current inhibition, and apoptosis-related protein expression regulation. These mechanisms were all dependent on bidentatide-induced inhibitory regulation of NR2B-containing NMDA receptors; thus, bidentatide may contribute to the development of neuroprotective agents that would likely possess the high selectivity and safety profiles inherent in peptide drugs.

Outcome and risk of ocular complications of managing children with chronic anterior uveitis with topical rimexolone 1.

PURPOSE: To investigate the efficacy and safety of 1% rimexolone ophthalmic suspension in children with chronic anterior uveitis under real-life conditions in a tertiary center. METHODS: This is a retrospective longitudinal study. Medical records were analyzed at baseline, 1, 3, 6 and 12 months before and after switching to rimexolone for best-corrected visual acuity (BCVA), oral steroid use, number of flares, IOP and anti-glaucoma management. RESULTS: Twenty-four patients (41 eyes) diagnosed with either anterior uveitis (n = 25, 60.0%) or panuveitis (n = 16, 40%) were enrolled. The mean age was 10.5 years (4-16 years). The number of patients requiring oral prednisolone reduced from 8 patients (32.0%) at baseline to 3 patients (20.0%) at 12 months (P < 0.001). Following baseline, the median number of uveitis flares reduced from 2.0 (inter-quartile range (IQR) 1.0-2.75) to 1.0 (IQR 0.0-1.0) compared to the 12 months before baseline (P < 0.001). The mean IOP reduced from baseline (22.0 ± 7.3 mmHg) to 1 month (18.8 ± 8.7 mmHg, P = 0.01) and remained stable up to 12 months (15.9 ± 5.0 mmHg, P < 0.001). Average BCVA, dose of oral prednisolone and anti-glaucoma treatments did not change compared to the baseline. The development for IOP ≥ 30 mmHg was associated with a known corticosteroid response [odds ratio (OR) 6.8, P = 0.003] and a dose > 7.5 mg/day oral prednisolone (OR 4.4, P = 0.033). CONCLUSIONS: Rimexolone 1% ophthalmic suspension is an effective and safe topical steroid for pediatric anterior uveitis.

Stress Impacts the Regulation Neuropeptides in the Rat Hippocampus and Prefrontal Cortex.

Adverse life experiences increase the lifetime risk to several stress-related psychopathologies, such as anxiety or depressive-like symptoms following stress in adulthood. However, the neurochemical modulations triggered by stress have not been fully characterized. Neuropeptides play an important role as signaling molecules that contribute to physiological regulation and have been linked to neurological and psychiatric diseases. However, little is known about the influence of stress on neuropeptide regulation in the brain. Here, we have performed an exploratory study of how neuropeptide expression at adulthood is modulated by experiencing a period of multiple stressful experiences. We have targeted hippocampus and prefrontal cortex (PFC) brain areas, which have previously been shown to be modulated by stressors, employing a targeted liquid chromatography-mass spectrometry (LC-MS) based approach that permits broad peptide coverage with high sensitivity. We found that in the hippocampus, Met-enkephalin, Met-enkephalin-Arg-Phe, and Met-enkephalin-Arg-Gly-Leu were upregulated, while Leu-enkephalin and Little SAAS were downregulated after stress. In the PFC area, Met-enkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, peptide PHI-27, somatostatin-28 (AA1-12), and Little SAAS were all downregulated. This systematic evaluation of neuropeptide alterations in the hippocampus and PFC suggests that stressors impact neuropeptides and that neuropeptide regulation is brain-area specific. These findings suggest several potential peptide candidates, which warrant further investigations in terms of correlation with depression-associated behaviors.

Separation and identification of mouse brain tissue microproteins using top-down method with high resolution nanocapillary liquid chromatography mass spectrometry.

Microproteins and endogenous peptides in the brain contain important substances that have critical roles in diverse biological processes, contributing to signal transduction and intercellular signaling. However, variability in their physical or chemical characteristics, such as molecule size, hydrophobicity, and charge states, complicate the simultaneous analysis of these compounds, although this would be highly beneficial for the field of neuroscience research. Here, we present a top-down analytical method for simultaneous analysis of microproteins and endogenous peptides using high-resolution nanocapillary LC-MS/MS. This method is detergent-free and digestion-free, which allows for extracting and preserving intact microproteins and peptides for direct LC-MS analysis. Both higher energy collision dissociation and electron-transfer dissociation fragmentations were used in the LC-MS analysis to increase the identification rate, and bioinformatics tools ProteinGoggle and PEAKS Studio software were utilized for database search. In total, we identified 471 microproteins containing 736 proteoforms, including brain-derived neurotrophic factor and a number of fibroblast growth factors. In addition, we identified 599 peptides containing 151 known or potential neuropeptides such as somatostatin-28 and neuropeptide Y. Our approach bridges the gap for the characterization of brain microproteins and peptides, which permits quantification of a diversity of signaling molecules for biomarker discovery or therapy diagnosis in the future.

N-Butyl-4-hydroxy-1,8-naphthalimide: A new matrix for small molecule analysis by matrix-assisted laser desorption/ionization mass spectrometry.

RATIONALE: The matrix plays an essential role in defining detection limits and ionization yields of analytes in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Small molecule MALDI-MS analyses commonly suffer from the high background interference generated from matrices. Moreover, the inhomogeneous crystallization of some matrices, such as 2,5-dihydroxybenzoic acid (DHB), is to the detriment of the quality or repeatability of detection. We have found that N-butyl-4-hydroxy-1,8-naphthalimide (BHN) can provide improved performance as a matrix for small molecule analysis. METHODS: BHN was evaluated in the low-mass region for its ionization efficiency, repeatability and background interference using O-acetyl-L-carnitine hydrochloride, Aß35-40, Aß35-42, and oxytocin as the model analytes. In addition, the modification effects of BHN on DHB were investigated for the in situ analysis of endogenous compounds in rat brain slices using Fourier transform ion cyclotron resonance (FTICR)-MS. RESULTS: BHN is capable of ionizing small molecules, including O-acetyl-L-carnitine hydrochloride and peptides, with high repeatability and low background interference signals. A low concentration of BHN (3 mM) modifies the crystallization state of DHB but still retains its ionization performance. The determination of small molecules desorbed from tissue slices was significantly improved by using a binary matrix of DHB and BHN, yielding superior signal-to-noise ratio and signal intensities. CONCLUSIONS: The new matrix BHN has exhibited suitability for the analysis of small molecules. Compared with the conventional matrices, CHCA and DHB, BHN provides a clean background in the low-mass region. In combination with DHB, the ability of BHN to form highly homogenous crystalline particles shows the clear beneficial effects of BHN for the reproducibility of MS detection.

Extensive characterization of peptides from Panax ginseng C. A. Meyer using mass spectrometric approach.

Panax ginseng is an important herb that has clear effects on the treatment of diverse diseases. Until now, the natural peptide constitution of this herb remains unclear. Here, we conduct an extensive characterization of Ginseng peptidome using MS-based data mining and sequencing. The screen on the charge states of precursor ions indicated that Ginseng is a peptide-rich herb in comparison of a number of commonly used herbs. The Ginseng peptides were then extracted and submitted to nano-LC-MS/MS analysis using different fragmentation modes, including CID, high-energy collisional dissociation, and electron transfer dissociation. Further database search and de novo sequencing allowed the identification of total 308 peptides, some of which might have important biological activities. This study illustrates the abundance and sequences of endogenous Ginseng peptides, thus providing the information of more candidates for the screening of active compounds for future biological research and drug discovery studies.