Anionic Proteins Research Articles

Quantum mechanical/molecular mechanical trajectory surface hopping molecular dynamics simulation by spin-flip time-dependent density functional theory.

This paper presents the nonadiabatic molecular dynamics simulation in the solution phase using the spin-flip time-dependent density functional theory (SF-TDDFT). Despite the single-reference level of theory, the SF-TDDFT method can generate the correct topology of S0/S1 crossing points, thus providing a natural S1 → S0 nonadiabatic transition. We extend the gas-phase trajectory surface hopping simulation with the SF-TDDFT [N. Minezawa and T. Nakajima, J. Chem. Phys. 150, 204120 (2019)] to the hybrid quantum mechanical/molecular mechanics (QM/MM) scheme. To this end, we modify the code to evaluate the electrostatic interaction between the QM and MM atoms and to extract the classical MM energy and forces from the MM program package. We apply the proposed method to the photoisomerization reaction of aqueous E-azomethane and anionic green fluorescent protein chromophore in water and compare the results with those of the previous simulation studies based on the multireference methods.

Compaction and self-association of megabase-sized chromatin are induced by anionic protein crowding.

Highly compacted chromatin, a complex of DNA with cationic histone proteins, is found in the nucleus of eukaryotic cells in an environment with a high concentration of macromolecular species, many of which possess a negative charge. In the majority of previous studies, however, these crowding conditions were experimentally modelled using neutral synthetic macromolecules such as polyethylene glycol (PEG). Despite the importance of the crowding agent charge in the condensation process of chromatin, to the best of our knowledge, the behavior of chromatin under conditions of anionic protein crowding has not been studied. Here, compaction of nearly megabase-long chromatin in the presence of the anionic globular protein BSA was investigated by single-molecule fluorescent microscopy (FM). We demonstrate different effects of anionic macromolecular crowders (MMCs) on DNA and chromatin, compared to neutral MMCs. While DNA molecules undergo gradual compaction into a globular form in the presence of ca. 20% w/v of BSA, chromatin fibres complete coil to globule transition at a much lower concentration of BSA (ca. 5% w/v). Furthermore, at higher concentrations of BSA in solution (>5% w/v), chromatin fibres self-associate and form large spherical or fibrillar supramolecular microstructures characterized by a high colloidal stability and dynamic intermolecular fluctuations. Formation of such self-organized colloids from chromatin is universal and characteristic of chromatin fibres of various lengths. Our results highlight the hitherto underappreciated effect of anionic MMC environment on chromatin higher-order structures that may play an important role in self-organization of chromatin in vivo.

Semi-permanent cationic coating for protein separations

Capillary electrophoresis has been used extensively for protein separations, but interactions of proteins with the negative charge on the surface of fused silica capillary create band broadening and diminish the separation efficiency. Coatings developed to mask the negative charge of the capillary affect the electroosmotic flow. The method presented in this work addresses these concerns through the use of a two-layer coating of a semi-permanent phospholipid substrate and cetyltrimethylammonium bromide (CTAB). When used alone, phospholipid coating suppresses the electroosmotic flow but cannot be used to simultaneously separate anionic and cationic proteins. When used alone, CTAB creates a dynamic coating that facilitates the separation of cationic proteins with good efficiency, but reduces the separation efficiency of anionic proteins. The use of a hybrid phospholipid-CTAB surface coating alleviates protein adsorption, as demonstrated through a comparison of protein separations obtained with a bare fused silica capillary. The hybrid phospholipid-CTAB surface enables high efficiency separations of cationic and anionic proteins simultaneously. This work verifies the role of the hydrophobic tail of CTAB in developing a stable coating with an electroosmotic flow of 3.14 × 10−4 cm2V-1s-1 (n = 10) from the cathode to the anode at a pH of 7. The coating yields a stable electroosmotic flow even after 2 h of flushing with background electrolyte devoid of CTAB (n = 3) and six consecutive protein injections with no flush sequence between runs. The coating can be used with background electrolytes with pH values ranging from 4 to 8 while maintaining 1% RSD (n = 10) in the electroosmotic flow for each background electrolyte. Six model proteins, lysozyme, ribonuclease A, α-chymotrypsinogen A, enolase, transferrin, and α-1-antitrypsin, with pI values ranging from 4.4 to 11 were used to demonstrate the stability of the phospholipid-CTAB coating, the lack of protein interaction with the wall, and the utility of the coating for the separation of proteins of similar isoelectric points and of protein isoforms.

An anionic human protein mediates cationic liposome delivery of genome editing proteins into mammalian cells

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (–30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (–30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (–30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.

Ordering Transitions in Liquid Crystals Triggered by Bioactive Cyclic Amphiphiles: Potential Application in Label-Free Detection of Amyloidogenic Peptides

We report the orientational behavior of nematic liquid crystals (LCs) influenced by a cyclic lipopeptide, polymyxin B (PmB). It was found that PmB can spontaneously self-assemble at aqueous-LC interfaces and induce a homeotropic ordering of the LCs at those interfaces, thus resulting in dark optical appearance of the LC under cross polarizers. Density functional theory studies substantiate the experimental findings that the stability of homeotropic anchoring of the LC is strongly influenced by the hydrophobic interactions between aliphatic tails of PmB and LC molecules along with the additional supramolecular interactions between their head groups. Interestingly, exposure of the PmB-laden aqueous-LC interface to anionic serum proteins such as bovine serum albumin (BSA) and human hemoglobin triggered a planar reorientation of the LC, leading to a bright optical state of the LC. This allows label-free characterization of the biomolecular interactions between proteins and antibiotics (i.e., PmB) in vitro at ...

Protein triggered ordering transitions in poly (L-lysine)-coated liquid crystal emulsion droplets

ABSTRACTHere, we report a simple and label-free methodology for real-time monitoring of adsorption of proteins such as bovine serum albumin (BSA), concanavalin A (ConA) (a lectin) and cathepsin D (CathD) (a tumour marker) on micrometer-sized poly (L-lysine) (PLL) functionalised liquid crystal (LC) droplets dispersed in aqueous phases. Earlier, we had demonstrated that PLL, a positively charged natural peptide, can induce homeotropic ordering of LCs at LC-aqueous interface, and thus PLL-adsorbed LC droplets showed radial director configuration. Herein, it was observed that subsequent non-specific adsorption of anionic proteins such as BSA, ConA and CathD can trigger a quick transition in director configuration of PLL-LC droplets (primarily dominated by electrostatic interactions) to pre-radial or bipolar, thus acting as a simple optical probe for detection of these proteins up to μg/mL of concentrations. Further, the detection limits for these proteins are found to vary (BSA<ConA<CathD) which follow the similar order as their anionic charges, thus suggesting the role of different binding affinities of protein-PLL in realising the director configuration of LC droplets. Overall, this study offers new pathways utilising ordering transition in LC droplets which will strengthen the principles to recognise biomolecular interactions for various interfacial and sensing applications.

Assessment of Functionals for TDDFT Calculations of One- and Two-Photon Absorption Properties of Neutral and Anionic Fluorescent Proteins Chromophores

Performance of DFT functionals with different percentages of exact Hartree-Fock exchange energy (EX) is assessed for recovery of the CC2 reference one- (OPA) and two-photon absorption (TPA) spectra of fluorescent proteins chromophores in vacuo. The investigated DFT functionals, together with their EX contributions are BLYP (0%), B3LYP (20%), B1LYP (25%), BHandHLYP (50%), and CAM-B3LYP (19% at short range and 65% at long range). Our test set consists of anionic and neutral chromophores as naturally occurring in the fluorescent proteins. For the first time, we compare TDDFT and CC2 methods for higher excited states than the S1 state, exhibiting relatively large TPA intensity. Our TDDFT results for neutral chromophores reveal an increase in excitation energies as well as TPA and OPA intensities errors, compared to CC2-derived results, as the DFT functional contains less exact exchange. The long-range-corrected CAM-B3LYP functional performs the best, closely followed by BHandHLYP, while BLYP usually significantly underestimates all investigated spectral properties, hence being the worst in reproducing the reference CC2 results. The hybrid B3LYP and B1LYP functionals can be roughly placed in between. We propose that TDDFT may underestimate the TPA intensities for neutral chromophores of fluorescent proteins due to underestimated oscillator strengths between some excited states. In the case of anionic chromophores, we find that B3LYP and B1LYP functionals overcome others in terms of reproducing CC2 excitation energies. On the other hand, however, TPA intensity is usually significantly underestimated, and in this respect, CAM-B3LYP functional seems to be again superior. In contrast to the case of neutral chromophores, it seems that a large magnitude of excited-state dipole moments or changes in dipole moments upon excitation may be the driving force behind high TPA transition moments.

Anticardiolipin (aCL) in sera from periodontitis subjects activate Toll-like receptor 4 (TLR4)

Anticardiolipin antibodies (aCL) have been reported to be present in 15–20% of sera from subjects with periodontitis at concentrations exceeding those found in 95% of the healthy adult population. These antibodies, albeit at concentrations exceeding those generally found in periodontitis subjects, are typically present in patients with the antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. aCL from APS patients are proinflammatory and can activate trophoblasts, macrophages, and platelets via cell-surface interactions with their target antigen beta-2-glycoprotein-I (β2GPI). β2GPI is an anionic phospholipid-binding serum protein that can associate with toll-like receptors (TLR’s) on the cell-surface, leading to cell activation following interaction with autoimmune aCL. We examined an expanded series of 629 sera from clinically characterized subjects for aCL content, and observed that 14–19% of these sera contained elevated (>95th %-tile) levels of aCL. We purified IgG from 16 subjects with elevated or normal levels of aCL and examined their ability to activate TLR2- or TLR4-transfected human embryonic kidney (HEK) cells, and observed that IgG from periodontitis patients with elevated aCL activated HEK-TLR4 cells, but not HEK-TLR2 cells. Prior removal of aCL by immunoabsorption significantly reduced the ability of IgG preparations from these sera to activate TLR4. Further experiments using a human first trimester trophoblastic cell line (HTR8 sv/neo) revealed that aCL from periodontitis patients stimulated IL-8 production, which was profoundly decreased if aCL was removed by immunoabsorption or if HTR8 sv/neo were pretreated with blocking anti-TLR4 antibodies. Thus, it appears that aCL from periodontitis patients can be proinflammatory, activating cells via TLR4. Since these antibodies are likely produced via molecular mimicry due to similarities between oral bacterial antigens and β2GPI, the data indicate that circulating serum aCL may induce or influence inflammatory responses at sites distant from the oral cavity.

Design of Hollow Protein Nanoparticles with Modifiable Interior and Exterior Surfaces

Protein-based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously assembles from a designed fusion protein subunit based on the geometric features of polyhedra. We show that TIP60 tolerates mutation and both its interior and exterior surfaces can be chemically modified. Moreover, TIP60 forms larger structures upon the addition of a cationic protein. Therefore, TIP60 can be used as a modifiable nano-building block for further molecular assembly.

Design of Hollow Protein Nanoparticles with Modifiable Interior and Exterior Surfaces

AbstractProtein‐based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously assembles from a designed fusion protein subunit based on the geometric features of polyhedra. We show that TIP60 tolerates mutation and both its interior and exterior surfaces can be chemically modified. Moreover, TIP60 forms larger structures upon the addition of a cationic protein. Therefore, TIP60 can be used as a modifiable nano‐building block for further molecular assembly.

New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors.

Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein–protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.

Immunomodulatory Effects of the Water-soluble Lectin from Moringa oleifera Seeds (WSMoL) on Human Peripheral Blood Mononuclear Cells (PBMC).

Moringa oleifera is used in traditional medicine as well as in food, cosmetic, and pharmaceutical industries. Water-soluble M. oleifera lectin (WSMoL) is an anionic protein isolated from the seeds of this tree. Until now, immune responses promoted by this lectin in human PBMC have not been investigated. The main objective of this study was to investigate the immunomodulatory effects of WSMoL on human PBMC through measurement of lymphocytes subsets, cytokine and nitric oxide levels. Human peripheral blood mononuclear cells (PBMC) were isolated through Ficoll technique, were incubated with WSMoL (10 µg/mL) for 24, 48 and 72 hours, and was performed immunophenotyping assay of lymphocytes and monocytes. Culture supernatants were used to determined cytokine and nitric oxide levels. Assays with cells subsets and cytokine production were performed through cytometry. Nitric oxide release assay was determinate by spectrophotometry. WSMoL induced the release of the cytokines TNF-α, IL-2, IL-6, IL-10 as well as nitric oxide. Incubation of PBMC with this lectin also led to activation of CD8+ T lymphocytes. WSMoL promotes immunomodulation in human PBMC inducing a potential wound healing profile and, in future in vivo assays, can be evaluated as adjuvant in immunosuppressive diseases and wound repair.

In silico investigation of the molecular effects caused by R123H variant in secretory phospholipase A2-IIA associated with ARDS

Phospholipase A2-IIA catalyzes the hydrolysis of the sn-2 ester of glycerophospholipids. A rare c.428G > A (p.Arg143His) variant in PLA2G2A gene was found in two infants affected by acute respiratory distress syndrome (ARDS) by whole coding region and exon/intron boundaries sequencing. To obtain insights into the possible molecular effects of the rare R123H mutation in secretory PLA2-IIA (sPLA2-IIA), molecular modelling, molecular dynamics (MD) using principal component analysis (PCA) and continuum electrostatic calculations were conducted on the crystal structure of the wild type protein and on a generated model structure of the R123H mutant. Analysis of MD trajectories indicate that the overall stability of the protein is not affected by this mutation but nevertheless the catalytically crucial H-bond between Tyr51 and Asp91 as well as main electrostatic interactions in the region close to the mutation site are altered. PCA results indicate that the R123H replacement alter the internal molecular motions of the enzyme and that collective motions are increased. Electrostatic surface potential studies suggest that after mutation the interfacial binding to anionic phospholipid membranes and anionic proteins may be changed. The strengthening of electrostatic interactions may be propagated into the active site region thus potentially affecting the substrate recognition and enzymatic activity. Our findings provide the basis for further investigation and advances our understanding of the effects of mutations on sPLA2 structure and function.

A Mathematical Description of Heat Transfer and a Procedure for Determining Temperature Characteristics of Electro-Ultrafiltration Process for Separating Industrial Solutions

A mathematical description of heat transfer is presented and a procedure is developed for calculating the temperature characteristics of electro-ultrafiltration process for separating industrial solutions in flatchamber type of apparatuses. The permeate and retentate temperatures, solution heating temperature, heating power, and amount of heat liberated in the process of separation of industrial solutions and effluents containing anionic surfactants, proteins, and phosphatic and biochemical substances are calculated by the developed procedure.

The effect of emulsifier type on the formation and stability of nanoemulsion gels

SummaryLiquid nanoemulsions are shown to transform into viscoelastic gels by reducing droplet size, increasing interfacial repulsive barrier between the nanodroplets and therefore increasing the effective oil volume fraction. The repulsive gelation in nanoemulsions can be achieved at a significantly lower oil volume fraction compared to conventional emulsion gels, making the nanoemulsion gel an attractive material for various low‐fat food applications. Gelation in nanoemulsions stabilized by anionic small molecule emulsifier and polymeric protein are compared in terms of gel strength, average droplet size, effective oil volume fraction, and long‐term gel stability. It is expected that higher stability and large surface area of nanoscale droplet size can further extend the application of nanoemulsion gels in the field of functional foods, cosmetics and pharmaceuticals.

Characterization of a water-soluble chitosan derivative and its potential for submucosal injection in endoscopic techniques

To examine the potential of chitosan-based agents for submucosal injection in endoscopic techniques, a chitosan derivative was prepared with lactose moieties linked to the amino groups of its glucosamine units (CH-LA). After dissolving CH-LA in neutral pH solutions, including physiological saline (CH-LA-S), its response to different concentrations of anionic glycosaminoglycans and proteins in the surrounding environment was examined. The CH-LA-S form changed in the presence of sulfated glycosaminoglycans (heparin, chondroitin sulfate, and mucin) and protein (fibrinogen). High concentrations of sulfated substrates in the solution caused the formation of larger structures. In contrast, in the presence of hyaluronan, 30mg/mL CH-LA-S did not form any large structures. Submucosal injection of 30mg/mL CH-LA-S into extracted swine stomachs showed a strong lifting effect of the gastric mucosa. These results indicate the potential utility of CH-LA-S as a submucosal injection for endoscopic techniques such as endoscopic submucosal dissection and mucosal resection of tumors.

Salt Triggers the Simple Coacervation of an Underwater Adhesive When Cations Meet Aromatic π Electrons in Seawater

Adhesive systems in many marine organisms are postulated to form complex coacervates (liquid-liquid phase separation) through a process involving oppositely charged polyelectrolytes. Despite this ubiquitous speculation, most well-characterized mussel adhesive proteins are cationic and polyphenolic, and the pursuit of the negatively charged proteins required for bulk complex coacervation formation internally remains elusive. In this study, we provide a clue for unraveling this paradox by showing the bulky fluid/fluid separation of a single cationic recombinant mussel foot protein, rmfp-1, with no additional anionic proteins or artificial molecules, that is triggered by a strong cation-π interaction in natural seawater conditions. With the similar condition of salt concentration at seawater level (>0.7 M), the electrostatic repulsion between positively charged residues of mfp-1 is screened significantly, whereas the strong cation-π interaction remains unaffected, which leads to the macroscopic phase separation (i.e., bulky coacervate formation). The single polyelectrolyte coacervate shows interesting mechanical properties including low friction, which facilitates the secretion process of the mussel. Our findings reveal that the cation-π interaction modulated by salt is a key mechanism in the mussel adhesion process, providing new insights into the basic understanding of wet adhesion, self-assembly processes, and biological phenomena that are mediated by strong short-range attractive forces in water.

Enhancing physicochemical properties of emulsions by heteroaggregation of oppositely charged lactoferrin coated lutein droplets and whey protein isolate coated DHA droplets

The formation and physicochemical stability of mixed functional components (lutein & DHA) emulsions through heteroaggregation were studied. It was formed by controlled heteroaggregation of oppositely charged lutein and DHA droplets coated by cationic lactoferrin (LF) and anionic whey protein isolate (WPI), respectively. Heteroaggregation was induced by mixing the oppositely charged LF-lutein and WPI-DHA emulsions together at pH 6.0. Droplet size, zeta-potential, transmission-physical stability, microrheological behavior and microstructure of the heteroaggregates formed were measured as a function of LF-lutein to WPI-DHA droplet ratio. Lutein degradation and DHA oxidation by measurement of lipid hydroperoxides and thiobarbituric acid reactive substances were determined. Upon mixing the two types of bioactive compounds droplets together, it was found that the largest aggregates and highest physical stability occurred at a droplet ratio of 40% LF-lutein droplets to 60% WPI-DHA droplets. Heteroaggregates formation altered the microrheological properties of the mixed emulsions mainly by the special network structure of the droplets. When LF-coated lutein droplets ratios were more than 30% and less than 60%, the mixed emulsions exhibited distinct decreases in the Mean Square Displacement, which indicated that their limited scope of Brownian motion and stable structure. Mixed emulsions with LF-lutein/WPI-DHA droplets ratio of 4:6 exhibited Macroscopic Viscosity Index with 13 times and Elasticity Index with 3 times of magnitudes higher than the individual emulsions from which they were prepared. Compared with the WPI-DHA emulsion or LF-lutein emulsion, the oxidative stability of the heteroaggregate of LF-lutein/WPI-DHA emulsions was improved. Heteroaggregates formed by oppositely charged bioactive compounds droplets may be useful for creating specific food structures that lead to desirable physicochemical properties, such as microrheological property, physical and chemical stabilities.

Screening, Purification and Characterization of Anionic Antimicrobial Proteins from Foeniculum Vulgare.

Foeniculum vulgare Mill., commonly called fennel, is a medicinal plant belonging to the Umbelliferae (Apiaceae) family, and is used in traditional medicine. Antibacterial peptides were isolated using sodium phosphate citrate buffer and, for extraction, cetyltrimethyl ammonium bromide (CTAB) buffer with pH 6, have been employed and antimicrobial activity tested against four reference strains. The extracted protein was subjected to 3 kDa dialysis and separation was carried out by DEAE-ion exchange chromatography and further proteins were identified by 2D gel electrophoresis. The results of Foeniculum vulgare elutes obtained from DEAE-ion exchange chromatography were tested for antibacterial activity. Elute 3 shows the highest antibacterial activity on Pseudomonas aeruginosa with a diameter of a zone of inhibition of 16 mm and IC50 value 25.02 (mcg/mL). Based on the findings of the wide usage in treatment of various ailments and day-to-day life, Foeniculum vulgare seeds were used in the present research and have shown promising antibacterial effects, which requires further proteomic research to authenticate the role of the anticipated proteins.

MP12-09 MATRIX PROTEIN DIFFERENCES BETWEEN URIC ACID AND CALCIUM OXALATE STONES

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology I1 Apr 2017MP12-09 MATRIX PROTEIN DIFFERENCES BETWEEN URIC ACID AND CALCIUM OXALATE STONES Jeffrey Wesson, Ann Kolbach-Mandel, Carley Davis, Neil Mandel, and Brian Hoffmann Jeffrey WessonJeffrey Wesson More articles by this author , Ann Kolbach-MandelAnn Kolbach-Mandel More articles by this author , Carley DavisCarley Davis More articles by this author , Neil MandelNeil Mandel More articles by this author , and Brian HoffmannBrian Hoffmann More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.431AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In general, kidney stones are crystal aggregates held together by an organic matrix, which contains mainly proteins. Little is known about possible differences in the protein distribution between different stone types that likely are critical to stone pathogenesis. Enrichment of highly anionic and highly cationic proteins was observed in calcium oxalate (CaOx) stone matrix compared to urine, suggesting a role for protein aggregation in this stone process. In this study, stone matrix proteins have been quantitatively identified in 3 archival uric acid (UA) stones, and compared to the distribution observed in 8 calcium oxalate stones using a previously reported method. METHODS Stone matrix proteins were isolated from 3 archival UA stone samples (>95% UA content) by dissolution in sequential washes with an EDTA/SDS solution at pH=8 with dithiothreitol added. The solubilized proteins were combined, then concentrated and desalted by ultradiafiltration. Proteomic analysis was performed at the Medical College of Wisconsin Innovation Center using non-labelled, quantitative mass spectrometry methods, including only proteins with 2 or more peptide matches at >85% confidence, after removing keratin and redundant proteins. RESULTS Strong protein signals (>3,000 SC per sample) were obtained from each UA matrix protein sample identifying 342 unique proteins. Of these, 180 proteins were found in both UA and CaOx stone matrix, accounting for 81% of total protein mass of the former and 94% of the latter. Albumin and uromodulin were found in both stone types, but at lower relative abundance than these proteins are found in urine, suggesting non-selective inclusion of these highly abundant urine proteins. Important differences were that none of the 5 predominant proteins in CaOx stone matrix was prominent in UA stone matrix, and highly anionic proteins were not enriched in UA stones. Highly cationic proteins were slightly more prominent in UA stone matrix than in CaOx stone former urine, but much less than observed in CaOx stone matrix. Nuclear proteins were much less strongly represented in UA stone matrix. Conversely, immunoglobulins and complement proteins were predominantly enriched in UA stone matrix and less prominent in CaOx stones. CONCLUSIONS Stone matrix proteins exhibit distinctly different patterns in UA compared to CaOx stones, implying different underlying pathogenesis. In particular, UA stone formation appears to be more dependent on inflammatory pathways and less dependent on cell injury processes and polyanion-polycation association compared to CaOx stone formation. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e148 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Jeffrey Wesson More articles by this author Ann Kolbach-Mandel More articles by this author Carley Davis More articles by this author Neil Mandel More articles by this author Brian Hoffmann More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...