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Tissue microenvironment properties like blood flow, extracellular matrix, or proximity to immune-infiltrate are important regulators of cell biology. However, methods to study regional protein expression in the native tissue environment are limited. To address this need, we developed a novel approach to visualize, purify, and measure proteins in situ using automated spatially targeted optical microproteomics (AutoSTOMP). Here, we report custom codes to specify regions of heterogeneity in a tissue section and UV-biotinylate proteins within those regions. We have developed liquid chromatography-mass spectrometry (LC-MS)/MS-compatible biochemistry to purify those proteins and label-free quantification methodology to determine protein enrichment in target cell types or structures relative to nontarget regions in the same sample. These tools were applied to (a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and (b) characterize inflammatory proteins enriched in IgG4+ lesions in human esophageal tissues. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools and sample availability are limited.

Spatially targeted optical microproteomics (STOMP) is a method to study region-specific protein complexity in primary cells and tissue samples. STOMP uses a confocal microscope to visualize structu...

Tissue microenvironment properties like blood flow, extracellular matrix or proximity to immune infiltrate are important regulators of cell biology. However, methods to study regional protein expression in context of the native tissue environment are limited. To address this need we have developed a novel approach to visualize, purify and measure proteins in situ using Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0. We previously implemented AutoSTOMP to identify proteins localized to the vacuoles of obligate intracellular microbes at the 1-2 μm scale within infected host cells1. Here we report custom codes in SikuliX to specify regions of heterogeneity in a tissue section and then biotin tag and identify proteins belonging to specific cell types or structures within those regions. To enrich biotinylated targets from fixed tissue samples we developed a biochemical protocol compatible with LC-MS. These tools were applied to a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and b) characterize inflammatory proteins enriched in IgG4+ lesions in esophageal tissue. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools are limited.

The selective vulnerability of hippocampal area CA1 to ischemia-induced injury is a well-known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen-glucose deprivation-reperfusion (OGD-RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain-derived neurotrophic factor (BDNF) or 7,8-dihydroxyflavone (7,8-DHF) exhibited reduced depression of the electrical activity triggered by OGD-RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD-RP. The protective effect of TrkB activation was limited to area CA3, as OGD-RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3-kinase/Akt (PI-3-K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD-RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase-3 activity in slices pretreated with BDNF and exposed to OGD-RP. We propose that PI-3-K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.

We aim at structure determining immunological machinery using a novel spatial selective proteomic tool, autoSTOMP, developed in Ewald lab. AutoSTOMP comprises of UV initiated biotin tagging by confocal microscopy, affinity isolation, and peptide identification by the mass spectrometer, rendering a precise characterization of micro-structures of interest (mSOIs) at intracellular organelle scale excluding surrounding area with the decent signal-to-noise ratio. Previously, autoSTOMP was employed to investigate the parasitophorous vacuole membrane, a unique intracellular membrane structure in apicomplexan infected cells. We broaden the application of autoSTOMP to respond to the calls from the research or clinical laboratory for a reliable, repeatable, and re-constructible protocol to explore the mSOIs of an animal specimen or a patient biopsy. We demonstrated, in the cardiac infarcted tissue in ischemic injured rat, the mSOIs stained against the macrophage/monocyte marker, CD68, are enriched in lysosomal proteins, and characteristic proteins involved in the Fcγ receptor-activated macrophage deformation, migration, and phagocytosis. Furthermore, in eosinophilic esophagitis (EoE) biopsy, we identify the lgG4+ cell-cluster structures are enriched in eosinophil-derived granule proteins and lgE receptor FcɛRI signaling associated proteins as well as food allergens to build up the correlation between patient’s diet with EoE progression.

Toxoplasma gondii is an obligate intracellular pathogen whose ability to grow and evade cell autonomous immunity depends on the parasitophorous vacuole membrane (PVM). The PVM forms from the host plasma membrane during invasion and is maintained by parasite effectors. Interferon-inducible GTPases (IIGs) are central in parasite clearance, however, the precise mechanism linking IIG attack of the PVM, parasite killing and inflammatory host cell death is not clear. To identify host and parasite proteins recruited to the PVM, we used Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP), a novel discovery technique that uses a confocal microscope to visualize and photo-label proteins for identification by LC-MS. Using IFNγ to induce IIG attack of the PVM and the TLR2 ligand Pam3CSK4 to upregulate inflammasome components we examined protein recruitment to the PVM in BMDCs. AutoSTOMP identified over 300 proteins differentially enriched near the PVM of unprimed, TLR2, IFNγ and IFNγ+TLR2 treated BMDCs. To identify candidate regulators of parasite clearance, we selectively purified proteins on the PVMs coated with IIG protein GBP2. We identified the inflammasome components caspase-1 and ASC as well as the apoptotic caspases-3 and -6 on GBP2-coated PVM. This was consistent with the observation that IFNγ priming was sufficient for host cell death and parasite restriction. BMDC death was partially dependent on the Aim2, ASC, Caspase1/11 and gasdermin D, however, blocking the inflammasome didn’t impair parasite restriction. By contrast, iNOS inhibitor, was sufficient to partially rescue parasite growth. In summary, we report a new tool to study the PVM proteome and identify regulators of parasite clearance and host cell death.

Pyramidal cells and astrocytes have differential susceptibility to oxygen-glucose deprivation and reperfusion (OGD-RP). It is known that excessive reactive oxygen species (ROS) in mitochondria initiates the cell death, while glutathione (GSH) is one of the major defenses against ROS. Although it is known that astrocytes contain a higher concentration of GSH than neurons, and that astrocytes can provide neurons with GSH, we are unaware of a detailed and quantitative examination of the dynamic changes in the mitochondrial GSH system in the two cell types during OGD-RP. Here, we determined mitochondrial membrane potential and the degrees of oxidation of the mitochondrially targeted roGFP-based sensors for hydrogen peroxide (OxD(P)) and GSH (OxD(G)). We also developed a method to estimate the mitochondrial GSH (mGSH) concentration in single cells in the CA1 region of organotypic hippocampal slice cultures at several time-points during OGD-RP. We find that mitochondrial membrane potential drops in pyramidal cells during OGD while it is relatively stable in astrocytes. In both types of cell, the mitochondrial membrane potential decreases during RP. During OGD-RP, mitochondrial peroxide levels are the same. Astrocytic mGSH is more than four times higher than in pyramidal cells (3.2 vs 0.7 mM). Astrocytic mGSH is drained from mitochondria during OGD, whereas in pyramidal cells it remains fairly constant. OxD(GSH) prior to and during OGD is lower (less oxidized) in pyramidal cells than astrocytes but the two nearly converge during RP. The larger changes of redox status in the GSH system in pyramidal cells than astrocytes is an upstream sign of the higher mortality of the pyramidal cells after facing an insult. The pattern of [mGSH] changes in the two cell types could be recognized as another mechanism by which astrocytes protect neurons from transient, extreme conditions.

A redox-sensitive Grx1-roGFP2 fusion protein was introduced by transfection into single pyramidal neurons in the CA1 subfield of organotypic hippocampal slice cultures (OHSCs). We assessed changes in the GSH system in neuronal cytoplasm and mitochondria during oxygen-glucose deprivation and reperfusion (OGD/RP), an in vitro model of stroke. Pyramidal cells in a narrow range of depths below the surface of the OHSC were transfected by gene gun or single-cell electroporation with cyto- or mito-Grx1-roGFP2. To mimic the conditions of acute stroke, we developed an optimized superfusion system with the capability of rapid and reproducible exchange of the solution bathing the OHSCs. Measurements of pO2 as a function of tissue depth show that in the region containing the transfected cells, the pO2 is well-controlled. We also found that the pO2 changes on the same time scale as changes in intracranial pressure, cerebral blood flow, and pO2 during acute stroke. Determining the reduction potential, EGSH, from the ratiometric fluorescence signal requires an absolute intensity measurement during calibration of the Grx1-roGFP2. Using the signal from cotransfected tdTomato as an internal standard during calibration improves quantitative measurements of Grx1-roGFP2 redox status and allows EGSH to be determined. EGSH becomes more reducing during OGD and more oxidizing during RP in mitochondria while changes in cytoplasm are not significant compared with controls.

Pittcon 2015 , held in New Orleans, LA, USA, 8–12 March 2015, covered the field of analytical chemistry and its applications broadly and deeply. This review covers only a very small portion of the program. It focuses on the interests of the authors.

The conformations and motions of lipid molecules under different membrane curvatures have important implications for transmembrane protein function, binding events, and overall membrane organization. This work reports on the local order parameters of saturated lipid molecules, as measured by (13)C NMR relaxation, under several curvature conditions to probe structural changes as a function of lipid bilayer curvature. Different curvature conditions are created by depositing phosphatidylcholine membranes on spherical beads of various diameters. The findings reveal that the order parameters are not a continuous function of the membrane curvature. While small (30 nm) and large (110 nm) diameter bilayers exhibit similar order parameters, bilayers with curvatures of 60-80 nm diameter show a consistently increased order parameter along the entire lipid molecule, indicating a higher packing density and lateral tension. Order parameters for curvatures between 60 and 80 nm also show molecular evidence for interdigitation.

Aims: The susceptibility of CA1 over CA3 to damage from cerebral ischemia may be related to the differences in reactive oxygen species (ROS) production/removal between the two hippocampal subfields. We aimed to measure CA1/CA3 differences in net ROS production in real time in the first 30 min of reperfusion in pyramidal cells. We aimed to determine the underlying cause of the differential vulnerability of CA1 and CA3. Results: Real-time determinations of mitochondrial H2O2 and, independently, glutathione (GSH) redox status from roGFP-based probes in individual pyramidal cells in organotypic hippocampal cultures during oxygen–glucose deprivation (OGD)–reperfusion (RP) demonstrate a significantly more oxidizing environment during RP in CA1 than CA3 mitochondria. Protein levels (immunohistochemistry and Western blots), roGFP2-based probe measurements during controlled mitochondrial production of ROS, and thioredoxin reductase (TrxR) inhibition by auranofin are consistent with a more effective mitoch...

Composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH sensitivity were developed for oral delivery of protein drugs, using bovine serum albumin (BSA) as a model drug. The composite drug-loaded microparticles with a mean particle size less than 200mum were prepared by a convenient shredding method. Since the microparticles were formed by tripolyphosphate cross-linking, electrostatic complexation by alginate and/or pectin, as well as ionotropic gelation with calcium ions, the microparticles exhibited an improved pH-sensitive drug release property. The in vitro drug release behaviors of the microparticles were studied in simulated gastric (pH 1.2 and pH 5.0), intestinal (pH 7.4) and colonic (pH 6.0 and pH 6.8 with enzyme) media. For the composite microparticles with suitable compositions, the releases of BSA at pH 1.2 and pH 5.0 could be effectively sustained, while the releases at pH 7.4, pH 6.8 and pH 6.0 increased significantly, especially in the presence of pectinase. These results clearly suggested that the microparticles had potential for site-specific protein drug delivery through oral administration.

A new method was developed to fabricate nanospheres and vesicles as drug carriers. The drug-loaded nanospheres and vesicles were prepared by self-assembly of alginate in aqueous media containing Ca2+ and CO32− ions under very mild conditions. The preparation method did not involve any organic solvent and surfactant and could offer good control over the morphology and the size of self-assemblies. Through adjusting the preparation conditions, nanosized drug-delivery systems with different shapes, that is, nanospheres and vesicles, could be obtained. The morphologies of the drug-delivery systems were observed by transmission electron microscopy (TEM). 5-Fluorouracil (5-FU), an anticancer drug, was encapsulated in the nanospheres and vesicles, and in vitro drug release behavior was investigated. The effect of drug-loading content on the release was studied. The release of 5-FU could be effectively sustained from both drug-loaded nanospheres and vesicles because the presence of CaCO3 in the nanospheres/vesicle...