Ion Reflector Research Articles

Improvement of reflectron time-of-flight mass spectrometer for better convergence of ion beam

Reflectron time-of-flight mass spectrometer (RTOF-MS) is a powerful tool to achieve high resolution in mass analysis of various ions. As it is often supposed that analyte ions are generated in a small volume in the ion-extraction region, its performance is not optimized for ions spread over a large volume. Not only the mass resolution is degraded due to incomplete space and energy focusing, but also the ion beam is subject to significant divergence of ion trajectories after it is reflected from an ion reflector. Here we present a novel design of RTOF-MS equipped with an additional electrostatic lens in front of a conventional grid-less ion reflector for improving convergence of an ion beam. The performance is examined by mass analysis of Ag3+ ions extracted from a volume of 6.3 cm3, i.e., a cylinder with 20-mm length and 20-mm diameter. The improved convergence of an ion beam is demonstrated by a two-dimensional image of ions on the ion detector. Mass resolution is evaluated as well.

A New Approach to Increasing the Resolution of a Mass Spectrometer with Wedge-Shaped Reflectors

The paper describes the investigation of the ion-optical properties of a laser TOF mass spectrometer including two successively positioned wedge-shaped ion mirrors. Some specific properties of the configuration of ion trajectories near their reflection in the second ion reflector are found. The dependence of aberrations on ion energy acquired toothed shape for the resolution of the analyzer higher than 3000–5000. The approximation of the dependence gave a 15th degree polynomial. The calculation of polynomial coefficients showed a great contribution to the duration of ion packets for aberrations of higher order. The discovered features allowed us to suggest a way of the local correction of nearby trajectories in the total ion flux. By correcting the local motion of individual groups of ions, we could reduce temporary aberration to 1–1.6 ns, depending on ion energy. For the time of ion flight ~35 μs, such duration limits the resolution of the analyzer by a value not less than 10000. The real length of ion drift path was about 30 cm. The total overall sizes of the ionoptical system were ~24 × 19 × 5 cm.

Microcystin-LRDetectedinaLowMolecularWeight FractionfromaCrudeExtractofZoanthussociatus.

Cnidarian constitutes a great source of bioactive compounds. However, research involving peptides from organisms belonging to the order Zoanthidea has received very little attention, contrasting to the numerous studies of the order Actiniaria, from which hundreds of toxic peptides and proteins have been reported. In this work, we performed a mass spectrometry analysis of a low molecular weight (LMW) fraction previously reported as lethal to mice. The low molecular weight (LMW) fraction was obtained by gel filtration of a Zoanthus sociatus (order Zoanthidea) crude extract with a Sephadex G-50, and then analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) in positive ion reflector mode from m/z 700 to m/z 4000. Afterwards, some of the most intense and representative MS ions were fragmented by MS/MS with no significant results obtained by Protein Pilot protein identification software and the Mascot algorithm search. However, microcystin masses were detected by mass-matching against libraries of non-ribosomal peptide database (NORINE). Subsequent reversed-phase C18 HPLC (in isocratic elution mode) and mass spectrometry analyses corroborated the presence of the cyanotoxin Microcystin-LR (MC-LR). To the best of our knowledge, this finding constitutes the first report of MC-LR in Z. sociatus, and one of the few evidences of such cyanotoxin in cnidarians.

An increase in the order of spatiotemporal focusing in the time-of-flight mass spectrometer with nonuniform field in the ion-acceleration regions

An algorithm for the calculation of electrostatic field in an arbitrary region of acceleration in the mass reflector using the criterion of an increase in the spatiotemporal focusing is presented. Nonuniform accelerating field providing spatiotemporal focusing of the third and fourth orders in the presence of uniform field in the reflector is calculated for a mass reflector with ionization on the surface of target. The effect of the size of input window of detector on the resolution in the presence of nonuniform field in (i) ion source and (ii) ion source and reflector is considered.

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

Time-of-Flight Mass Spectrometer with Transaxial Ion Reflector

Two variants of application of a transaxial mirror with stigmatic spatial time-of-flight focusing in the time-of-flight mass spectrometer have been considered. In the first variant, the transaxial mirror is used as an ion reflector in the ordinary scheme of the time-of-flight mass reflectron. In the second variant, the transaxial mirror simultaneously fulfills the function of the ion reflector and corrector of aberrations caused by the energy spread of ions in the package, formed by the ion source of the mass reflectron. The expressions defining the conditions of stigmatic spatial time-of-flight focusing in the transaxial mirror and the combined system consisting of an ion source and a mirror have been derived. The relationships between geometrical and electrical parameters of three- and four-electrode transaxial mirrors realizing these conditions have been obtained by numerical calculations.

Design of Velocity Map Imaging Spectrometer Equipped with a Mass Gate Discriminating Particular Photofragments

A photoionization spectrometer for velocity map imaging has been developed for measuring the scattering distribution of fragment ions from polyatomic molecules. The spectrometer contains a mass gate and an ion reflector which are able to discriminate ions with a particular mass-to-charge ratio m/z. The basic functions and feasibility of these devices were tested experimentally and theoretically. First, the photoions from Kr and C60 were extracted into a time-of-flight (TOF) mass spectrometer by a transient or continuous electrostatic field. When the pulse application on the mass gate was tuned to the arrival timing of ions with a specific m/z, the peak of the selected ions alone was present on a TOF spectrum. Second, compatibility between velocity map imaging and ion discrimination was investigated by the computer simulations of the ion trajectories of photofragments from C60. A pulsed voltage was applied to the mass gate synchronously with the arrival timing of C58+ ions. The initial three-dimensional velocity distribution of C58+ was projected onto the image plane with an energy resolution better than 10 meV. The C58+ image was free from the contamination of other ions such as C60+ and C56+.

Physicochemical characterization of biosurfactant and its potential to remove oil from soil and cotton cloth

An alkaliphilic bacterium, Klebsiella sp. strain RJ-03, produced a biosurfactant, which showed low viscosity with pseudoplastic rheological behavior and exhibited emulsification activity with oils and hydrocarbons. The biosurfactant has excellent oil removing efficiency as compared to chemical surfactants. The isolated biosurfactant has compatibility with detergents and enhanced oil removing efficiency from soil and cotton cloths. It comprised of sugar, uronic acid, protein and sulfate. GC–MS analysis confirmed the presence of six monosaccharides (w/w), glucose (6.65%), galactose (23.98%), rhamnose (14.94%), mannose (17.54%), fucose (9.47%) and 6-O-Me-galactose (1.4%). It is a high molecular weight, thermostable biopolymer showing degradation above 300°C. Positive ion reflector mode of MALDI TOF-TOF MS analysis revealed series of low and mid range mass peaks (m/z) corresponding to mono-, di-, tri- and oligo-saccharides content. The NMR, FT-IR, EDX-SEM, AFM and PSD analysis confirmed the presence of functional groups, bonds, elements and particle size respectively.

Isolation and structural characterization of biosurfactant produced by an alkaliphilic bacterium Cronobacter sakazakii isolated from oil contaminated wastewater

Production of biosurfactant from an alkaliphilic bacterium Cronobacter sakazakii (accession no. JN398668) was screened by haemolytic assay, emulsifying activity and surface tension measurement. Biosurfactant, comprised of total sugars (73.3%), reducing sugars (1.464%), protein (11.9%), uronic acid (15.98%) and sulfate (6.015%), showed low viscosity with pseudoplastic rheological behavior and exhibited significant emulsification activity with oils and hydrocarbons. A series of low and mid range mass peaks (m/z) corresponding to mono-, di-, tri- and oligosaccharides were detected in the positive ion reflector mode of MALDI TOF–TOF MS. GC–MS analysis revealed composition of monosaccharide moieties (w/w) viz. glucose (14%), mannose (24%), galactose (14%), xylose (20%) and arabinose (1.9%). 1H NMR, FT-IR and EDX analyses confirmed the characteristic various functional groups, bonds and elements respectively. Thermostability (up to 260 °C) and CI (0.456) were determined by TG and DSC analyses. Inherent properties of biosurfactant make it a potential candidate for bioremediation of oil and hydrocarbons.

Isolation and characterization of exopolysaccharides from seaweed associated bacteria Bacillus licheniformis

In the present study, EPS secreted by the endophytic bacterium Bacillus licheniformis was isolated and characterized. The molecular masses of the EPS were 1540 and 44,565 kDa and 1H NMR, FT-IR and UV–Vis spectral analyses revealed prevalence of characteristic primary amine-group, aromatic-compound, halide and aliphatic alkyl-group in addition to Na, P, Ca, C, O, Cl and S as inferred from EDX analysis. XRD and DSC analysis confirmed the amorphous nature of EPS, showing an average particle size of 24.977 μm ( d 0.5) with 191 nm average roughness. The positive ion reflector mode of MALDI TOF-TOF MS exhibited a series of low and high mass peaks corresponding to various oligosaccharides and polysaccharides respectively. Further, GC–MS analysis revealed its four monosaccharide constituents glucose, galactose, mannose and arabinose. The potential heterogeneous properties of EPS as revealed in the present study may be explored in various biotechnological and industrial applications.

The Penicillium Chrysogenum Extracellular Proteome. Conversion from a Food-rotting Strain to a Versatile Cell Factory for White Biotechnology

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54-1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54-1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.

Characterization of extracellular polymeric substances produced by micro-algae Dunaliella salina

Extracellular polymeric substances comprised of average molecule size 1264.354 μm, exhibited characteristic diffraction peaks at 6.025°, 9.675°, 22.775° and 28.475° with d-spacing 14.74755, 9.36297, 3.88747 and 3.11512 Å, respectively. EDX confirms the presence of sulphate (2.7%) and 1H NMR reveals uronic acid, primary amine, aromatic-compounds, halides, aliphatic alkyl and sulfides. EPSs were thermostable upto 270 °C with CI xrd 0.12 and CI DSC 0.18. The dynamic viscosity is significantly high at pH 3.0 and decreases concomitantly with shear rate, confirming pseudoplastic rheological property. MALDI TOF–TOF represents a series of masses in linear mode corresponding to mass of pentose and hexose with ions. The positive ion reflector mode exhibited low mass peaks (m/z) corresponding to oligosaccharide and higher peaks for polysaccharide consist of different ratio of pentose and hexose associated with ions. EPSs allow further exploration of D. salina as potential EPSs producer and make it a promising candidate for biotechnological and industrial exploitation.

Proteomic Analysis of Penile Protein Alterations in a Rat Model of Cavernous Nerve Injury

Purpose: Cavernous nerve resection (CNR) in rats is a standard model of animal experiments on erectile dysfunction (ED) that occurs after radical prostatectomy (RP). Injured cavernous nerves after surgery can cause fibrosis and apoptosis that lead to penile structural changes that may be accompanied by alterations of protein expression. This study aimed to analyze the changes in protein after CNR in Wistar Kyoto rats. Materials and Methods: Using 8-week-old male Wistar Kyoto rats, sham and CNR operation under a microscope were performed. Two and 8 weeks after surgery, we applied 2-DE and MALDI-TOF/TOF (AB 4700) to identify differently expressed penile proteins after CNR. 2-DE gels were stained with silver nitrate and were analyzed with PDQuest. After in-gel digestion, peptide mass spectra were obtained by MALDI-TOF/TOF mass spectrometry in the positive ion reflector mode. The obtained data were screened with a rat database from both the NCBI and the Swiss-Prot/TrFMBL home page. Results: The proteins that were changed more than 1.5-fold compared with the sham group were annexin A4 and pyruvate kinase (PK). Annexin A4 was increased by 1.75-fold after 2 weeks, whereas PK was decreased by 4.16 after 8 weeks. These results were confirmed by immunohistochemistry. Conclusions: Annexin A4 in the CNR group was increased, which may be related to emiocytosis during apoptosis. The decrease in PK of the CNR group is assumed to be related to a decrease in efficacy during glycolysis. Further study will be needed to elucidate the molecular pathophysiology of ED after cavernous nerve injury. (Korean J Urol 2009;50:498-504) 󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏

A Generic Approach for the Purification of Signaling Complexes That Specifically Interact with the Carboxyl-terminal Domain of G Protein-coupled Receptors

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets. Recent progress has shown that GPCRs are part of large protein complexes that regulate their activity. We present here a generic approach for identification of these complexes that is based on the use of receptor subdomains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His6-tagged baits, immobilized metal affinity chromatography, one- and two-dimensional gel electrophoresis separation and mass spectrometric identification. The carboxyl-terminal tails (C-tails) of the human MT1 and MT2 melatonin receptors, two class A GPCRs, were used as models to purify protein complexes from mouse brain lysates. We identified 32 proteins that interacted with the C-tail of MT1, 14 proteins that interacted with the C-tail of MT2, and eight proteins that interacted with both C-tails. Several randomly selected proteins were validated by Western blotting, and the functional relevance of our data was further confirmed by showing the interaction between the full-length MT1 and the regulator of G protein signaling Z1 in transfected HEK 293 cells and native tissue. Taken together, we have established an integrated and generic purification strategy for the identification of high quality and functionally relevant GPCR-associated protein complexes that significantly widens the repertoire of available techniques.

Cosima – High Resolution Time-of-Flight Secondary Ion Mass Spectrometer for the Analysis of Cometary Dust Particles onboard Rosetta

The ESA mission Rosetta, launched on March 2nd, 2004, carries an instrument suite to the comet 67P/Churyumov-Gerasimenko. The COmetary Secondary Ion Mass Anaylzer – COSIMA – is one of three cometary dust analyzing instruments onboard Rosetta. COSIMA is based on the analytic measurement method of secondary ion mass spectrometry (SIMS). The experiment’s goal is in-situ analysis of the elemental composition (and isotopic composition of key elements) of cometary grains. The chemical characterization will include the main organic components, present homologous and functional groups, as well as the mineralogical and petrographical classification of the inorganic phases. All this analysis is closely related to the chemistry and history of the early solar system. COSIMA covers a mass range from 1 to 3500 amu with a mass resolution m/Δm @ 50% of 2000 at mass 100 amu. Cometary dust is collected on special, metal covered, targets, which are handled by a target manipulation unit. Once exposed to the cometary dust environment, the collected dust grains are located on the target by a microscopic camera. A pulsed primary indium ion beam (among other entities) releases secondary ions from the dust grains. These ions, either positive or negative, are selected and accelerated by electrical fields and travel a well-defined distance through a drift tube and an ion reflector. A microsphere plate with dedicated amplifier is used to detect the ions. The arrival times of the ions are digitized, and the mass spectra of the secondary ions are calculated from these time-of-flight spectra. Through the instrument commissioning, COSIMA took the very first SIMS spectra of the targets in space. COSIMA will be the first instrument applying the SIMS technique in-situ to cometary grain analysis as Rosetta approaches the comet 67P/Churyumov-Gerasimenko, after a long journey of 10 years, in 2014.

Unusual N-Glycan Structures in α-Mannosidase II/IIx Double Null Embryos Identified by a Systematic Glycomics Approach Based on Two-dimensional LC Mapping and Matrix-dependent Selective Fragmentation Method in MALDI-TOF/TOF Mass Spectrometry

alpha-Mannosidase IIx (MX) is an enzyme closely related to alpha-mannosidase II (MII), a key enzyme in N-glycan biosynthesis that catalyzes the first step in conversion of hybrid- to complex-type N-glycans in Golgi apparatus. Recently we generated MII/MX double knock-out mice and found that double nulls completely lack the complex-type N-glycans (Akama, T. O., Nakagawa, H., Wong, N. K., Sutton-Smith, M., Dell, A., Morris, H. R., Nakayama, J., Nishimura, S.-I., Pai, A., Moremen, K. W., Marth, J. D., and Fukuda, M. N. (2006) Essential and mutually compensatory roles of alpha-mannosidase II and alpha-mannosidase IIx in N-glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 8983-8988). In the present study, we determined minor but unusual N-glycan structures found in MII/MX double knock-out mice. We identified such N-glycans by a systematic glycomics approach applying a two-dimensional LC mapping database and matrix-dependent selective fragmentation technique in MALDI-TOF/TOF MS, a highly sensitive and reliable technique that provides specific fragmentations enabling the determination of precise oligosaccharide structures including regioisomers (Kurogochi, M., and Nishimura, S.-I. (2004) Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097-6101). Quantitative profiling of all N-glycan structures including minor components from MII/MX nulls, MII nulls, MX nulls, and wild-type mice at embryonic day 15.5 yielded a total of 37 species when structural heterogeneity was reduced by the removal of the sialic acids. Among six unusual N-glycan structures, two glycoforms were novel and were found only in MII/MX double nulls. We characterize such structure as pseudocomplex-type N-glycans. The present study demonstrated that use of the versatile matrix-dependent selective fragmentation method in MALDI-TOF/TOF MS greatly accelerates detailed structural analysis of a trace amount of N-glycans.

Redox Activation of Aldose Reductase in the Ischemic Heart

Aldose reductase (AR) reduces cytotoxic aldehydes and glutathione conjugates of aldehydes derived from lipid peroxidation. Its inhibition has been shown to increase oxidative injury and abolish the late phase of ischemic preconditioning. However, the mechanisms by which ischemia regulates AR activity remain unclear. Herein, we report that rat hearts subjected to ischemia, in situ or ex vivo, display a 2-4-fold increase in AR activity. The AR activity was not further enhanced by reperfusion. Activation increased Vmax of the enzyme without affecting the Km and decreased the sensitivity of the enzyme to inhibition by sorbinil. Enzyme activation could be prevented by pretreating the hearts with the radical scavenging thiol, N-(2-mercaptoproprionyl)glycine or the superoxide dismutase mimetic, Tiron, or by treating homogenates with dithiothreitol. In vitro, the recombinant enzyme was activated upon treatment with H2O2 and the activated, but not the native enzyme, formed a covalent adduct with the sulfenic acid-specific reagent dimedone. The enzyme activity in the ischemic, but not the nonischemic heart homogenates was inhibited by dimedone. Separation of proteins from hearts subjected to coronary occlusion by two-dimensional electrophoresis and subsequent matrix-assisted laser desorption ionization time-of-flight/mass spectrometry analysis revealed the formation of sulfenic acids at Cys-298 and Cys-303. These data indicate that reactive oxygen species formed in the ischemic heart activate AR by modifying its cysteine residues to sulfenic acids.

Release of Hypoacetylated and Trimethylated Histone H4 Is an Epigenetic Marker of Early Apoptosis

Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers.

Differential Requirement for Phospholipase D/Spo14 and Its Novel Interactor Sma1 for Regulation of Exocytotic Vesicle Fusion in Yeast Meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.

Blue Native/SDS-PAGE Analysis Reveals Reduced Expression of the mClCA3 Protein in Cystic Fibrosis Knock-out Mice

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.