Photodegradation Pathways of Protein Disulfides: Human Growth Hormone.

PURPOSE: Comprehensive product characterization was performed for the photodegradation of protein disulfides, representatively of human growth hormone (somatotropin; hGH), in order to provide a product database, which will be useful for the general analysis of protein stability. METHODS: HGH was photo-irradiated at λ = 254 and λ > 295 nm and tryptic digests were analyzed by HPLC-MS to investigate light-induced disulfide degradation pathways. RESULTS: A total of 60 products were detected, and structures/tentative structures were assigned to the products by MS2 and MS3 analysis. The main products were reduced Cys residues, dithiohemiacetal, thioether and disulfide scrambling products. In addition, we detected Cys degradation products such as Cys thioaldehyde, dehydroalanine (Dha), Ala, Ser semialdehyde, Ser, S-sulfocysteine, and Gly. Frequently, the tryptic fragments contained more than one modification, i.e. a Cys degradation product in close proximity to a dehydrated amino acid. Several novel cross-links were detected between Cys and Tyr, Cys, Ser and Phe, Cys and Trp, and Trp and Tyr. Photo-induced protein fragmentation was detected specifically at or in close proximity to the disulfide bond between T6 and T16. An in-house packed 75 cm nano-column enabled us to resolve various isomers/diastereomers of the photo-degradation products. CONCLUSION: A comprehensive analysis of photodegradation products revealed a variety of novel photo-products, including cross-links, originating from disulfide degradation. The mechanisms of product formation are discussed.

Profiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography-Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-µm Particles.

In recent years protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficult to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kspi range, accommodates the use of capillary columns in excess of one meter packed with sub-2 µm particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (λ 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at the higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.

Folate determination in human health: UPLC-MS/MS is the emerging methodology of choice.

This Perspective provides a brief description of the essential role that folates play in human health, together with an overview of the various analytical methods that have been used for quantitation of folates in human populations over the past few decades. Essentially, folate methodology has evolved from microbiological assay-based, to binding-based technology and, more recently, to separation-based methodology. Separation-based methods initially used traditional LC in conjunction with various detection techniques, with the most recent methods utilizing UPLC-MS/MS. Current UPLC methods offer exceptional speed, sensitivity and quantitation ability for the monoglutamate folate isoforms. It appears that the only limitation to properly quantifying all folates as polyglutamates, some 40-50 species, is the current lack of corresponding stable-isotope standards. Clearly, UPLC-MS/MS is emerging as the 'method of choice' for the determination of folates, whether in support of basic research, clinical investigations or population studies.

New single isomer negatively charged ß-cyclodextrin derivatives as chiral selectors in capillary electrophoresis.

To improve resolution power of chiral selector and enantiomeric peak efficiency in CE, single isomer negatively charged ß-CD derivatives, mono(6-deoxy-6-sulfoethylthio)-ß-CD (SET-ß-CD) bearing one negative charge and mono[6-deoxy-6-(6-sulfooxy-5,5-bis-sulfooxymethyl)hexylthio]-ß-CD (SMHT-ß-CD) carrying three negative charges, were synthesized. The structure of these two ß-CD derivatives was confirmed by (1)H NMR and MS. SET-ß-CD and SMHT-ß-CD successfully resolved the enantiomers of several basic model compounds. SMHT-ß-CD provided for a significantly greater enantioseparation than SET-ß-CD at lower concentrations. This appears to be due to the higher binding affinity of SMHT-ß-CD to the model compounds and the wider separation window resulting from an increased countercurrent mobility of the selector. Overall, the new chiral selectors provided enantioseparations with high peak efficiency while avoiding peak distortion due to polydispersive and electrodispersive effects. The information obtained from an apparent binding constant study suggested that the enantioseparation of the model compounds followed the predictions of charged resolving agent migration model and that the observed degree of enantioseparation difference were due to the magnitude of differences in both enantiomer-chiral selector binding affinities (ΔK) and the mobilities of the complexed enantiomers (Δµ(c)).

Comprehensive quantitative measurement of folate polyglutamates in human erythrocytes by ion pairing ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: The erythrocyte folate pool is reflective of an individual's long-term folate status; however, comprehensive quantitative determination of the various folate isoforms including polyglutamation (Glu(n)) status has posed an analytical problem. Factors complicating such analysis are the absence of authentic (isotope-labeled) standards and the large number of potential analytes. The present work presents high-throughput analytical methodology for the indirect comprehensive quantitation of the erythrocyte folate pool with commercially available standards. METHODS: The erythrocyte folate pool was determined comprehensively by utilizing a cascade of three complementary ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS) assays. In a first assay utilizing ion-pairing UPLC/MS/MS the relative (%) polyglutamation distribution (Glu(3-10)) of 5-methyltetrahydrofolate, tetrahydrofolate and 5-formyltetrahydrofolate is determined in a thermal extract obtained from packed erythrocytes, not requiring analytical standards. Quantitation of the erythrocyte folate pool was accomplished by performing two additional stable isotope dilution UPLC/MS/MS assays to determine whole blood and plasma folate content, utilizing commercially available [(13)C(5)]-labeled analogs of the Glu(1) analytes. Based on the values provided by each individual assay the comprehensive erythrocyte folate content could be calculated. RESULTS: The various assays have been validated for intra- and inter-run precision, accuracy, linearity and are robust. The method was sensitive enough to measure the comprehensive erythrocyte folate distribution in a Down's syndrome patient with extremely low folate, bearing the C677T mutation in the gene encoding for methylenetetrahydrofolate reductase. CONCLUSIONS: The erythrocyte folate pool can be comprehensively quantitated by running three complementary UPLC/MS/MS assays. The present assays are robust and allow for high-throughput analysis. The method can be utilized to support larger investigations that investigate the relationship between folate isoform and polyglutamation distribution and disease pathogenesis.

Measurement of methotrexate polyglutamates in human erythrocytes by ion-pair UPLC-MS/MS.

BACKGROUND: Low-dose methotrexate is used for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis, but its effectiveness greatly varies between individuals. Therapeutic drug monitoring of intracellular methotrexate metabolites, the γ-polyglutamates (MTXGlu(n)), in human erythrocytes has shown promise in providing a basis for individualization of therapy. RESULTS: This work presents expedient methodology for the analysis of MTXGlu(1-7) in human erythrocytes by ion-pair UPLC with detection by tandem MS (UPLC-ESI-MS/MS). The use of N,N-dimethylheptylamine as an ion-pair agent was found to be favorable over others. Thermal extraction of erythrocyte lysates provides a simple one-step extraction procedure. The entire chromatographic run time is 6 min and the assay was validated within the therapeutic range of these metabolites CONCLUSION: The developed sample preparation procedure in combination with ion-pair UPLC-ESI-MS/MS analysis allowed for expedient quantitation of MTXGlu(1-7) in human erythrocytes. The rapid analysis time would enable therapeutic drug monitoring of MTXGlu(1-7) in the clinic.

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides.

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO(2))LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.

LC-MS/MS method for the determination of carbamathione in human plasma.

Liquid chromatography-tandem mass spectrometry methodology is described for the determination of S-(N,N-diethylcarbamoyl)glutathione (carbamathione) in human plasma samples. Sample preparation consisted of a straightforward perchloric acid medicated protein precipitation, with the resulting supernatant containing the carbamathione (recovery ~98%). For optimized chromatography/mass spec detection a carbamathione analog, S-(N,N-di-i-propylcarbamoyl)glutathione, was synthesized and used as the internal standard. Carbamathione was found to be stable over the pH 1-8 region over the timeframe necessary for the various operations of the analytical method. Separation was accomplished via reversed-phase gradient elution chromatography with analyte elution and re-equilibration accomplished within 8 min. Calibration was established and validated over the concentration range of 0.5-50 nM, which is adequate to support clinical investigations. Intra- and inter-day accuracy and precision determined and found to be <4% and <10%, respectively. The methodology was utilized to demonstrate the carbamathione plasma-time profile of a human volunteer dosed with disulfiram (250 mg/d). Interestingly, an unknown but apparently related metabolite was observed with each human plasma sample analyzed.

Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration.

Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K(3)Fe(CN)(6) to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005-1 µM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 µM K(3)Fe(CN)(6) in 0.1 M Na(2)HPO(4) buffer (pH 9.0) was converted with yields >95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC-MS-MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 µg digested cell proteins, respectively. LC-MS-MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations.

Bioanalytical method development for a generation 5 polyamidoamine folic acid methotrexate conjugated nanoparticle.

Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9-N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 microL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.

A novel high-performance liquid chromatography/mass spectrometry method for improved selective and sensitive measurement of methotrexate polyglutamation status in human red blood cells.

The folate antagonist methotrexate is commonly used in low dose for treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Therapeutic effects are attributed to intracellular levels of various methotrexate polyglutamates. The present methodology, combining a simple preparation step with ion-pairing reversed-phase liquid chromatography and electrospray ionization mass spectrometry, is suitable for the measurement of methotrexate and its polyglutamates(2-7), in human red blood cells. Sample preparation consists of perchloric acid protein precipitation followed by solid-phase extraction. Baseline separation of all analytes was achieved within 10 min using a Phenomenex Synergy C18 column together with a gradient solvent program obtained from blending acetonitrile with pH 7.5, 5 mM aqueous dimethylhexylamine. Seven methotrexate polyglutamates were detected using multiple reaction monitoring, with the mass spectrometer operating in positive ion mode. Using 20 microL injection volumes, limits of detection were 2.5 nM for individual methotrexate polyglutamates, while large volume (100 microL) injections led to detection limits of 0.5 nM and linear calibration from 0.5 to 100 nM for individual analytes. Finally, the presented methodology was applied for the analysis of methotrexate and its polyglutamates in red blood cells obtained from patients being treated for juvenile idiopathic arthritis with methotrexate. Significantly, the methodology proved suitable for determination of long-chain methotrexate polyglutamates(5-7) and further, appears to be superior with respect to sensitivity, selectivity and speed as compared to all previously described approaches.

Selective fluorogenic derivatization of 3-nitrotyrosine and 3,4-dihydroxyphenylalanine in peptides: a method designed for quantitative proteomic analysis.

There is a need for the selective derivatization and enrichment of posttranslational protein modifications from tissue samples. This chapter describes a method for the selective derivatization of 3-nitrotyrosine (after reduction to 3-amino-tyrosine) and 3,4-dihydroxyphenylalanine with benzylamine derivatives to yield 6-amino- and 6-benzylamine-substituted benzoxazoles, which display characteristic fluorescence properties. The methodology can be expanded to other substituted benzylamines, which carry functional groups for affinity enrichment.

Synthesis of sulfonamide- and sulfonyl-phenylboronic acid-modified silica phases for boronate affinity chromatography at physiological pH.

Two new types of boronate affinity solid phases were synthesized and characterized. The materials were prepared by silylation of porous silica gel with monochlorosilane derivatives containing synthetic sulfonyl- and sulfonamide-substituted phenylboronic acids. The new solid phases were evaluated for boronate affinity chromatography with aryl and alkyl cis-diol compounds and were found to be suitable for the retention of cis-diols under acidic conditions. Significant correlations between the retention factor (K) and the pH of the mobile phase demonstrate that the binding of cis-diols to the solid phases is best rationalized by chelation. Based on the lower pKa, caused by the electron-withdrawing effects of the sulfonyl and sulfonamide groups, these media display an enhanced affinity for cis-diols as compared with unsubstituted phenylboronic acid. Using isocratic elution, a mixture of various biologically relevant l-tyrosines, l-DOPA, and several catecholamines were resolved with a mobile phase composed of 0.05M phosphate buffer (pH 5.5). Mono-, di-, and triphosphates of adenosine were also separated at pH 6.0. Hence, the new boronate solid phase offers efficient affinity separation and purification of cis-diol-containing molecules under rather mild pH conditions.

Amino acid and peptide analysis using derivatization with p-nitrophenol-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether and capillary electrophoresis with electrochemical detection.

The amine derivatization reagent p-nitrophenol-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether (NDTE) was used in conjunction with capillary electrophoresis (CE) and electrochemical detection (EC) for the pre-separation derivatization of primary amine analytes present in aqueous solution. Glycine, several dipeptides and angiotensin II were used as model analytes. A miniaturized EC detection cell was designed and fabricated, which featured a fractured-joint field decoupler with a fixed end-column carbon fiber electrode. When a series of glycine and angiotensin II calibration solutions were derivatized with NDTE followed by CE-EC determination, linear calibration plots resulted with pre-derivatization concentration limits of detection of 500 nM (106 attomoles on-column) and 6 microM (1.275 femtomoles on-column), respectively.

Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection.

Two analytical methods were developed for quantitative determination of DADLE (H(2)N-Tyr-D-Ala-Gly-Phe-D-Leu-COOH) and its two cyclic prodrugs in rat plasma. For high-performance liquid chromatography with fluorescence detection (LC-FLU), precolumn derivatization of DADLE was accomplished by labeling the N-terminal amino group with the reagent naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA/CN) to form a highly fluorescent 1-cyanobenz[f]isoindole (CBI) derivative. A multi-dimensional LC system was employed to improve selectivity, and solid-phase extraction (SPE) was used for plasma sample preparation. The cyclic prodrugs were converted to DADLE prior to their derivatization. With fluorescence detection after derivatization, the limit of quantitation (LOQ) was 6 ng ml(-1) for the analysis of DADLE, and good linearity was observed up to 6000 ng ml(-1) in rat plasma. Quantitative analysis of DADLE and its cyclic prodrugs was also performed using liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Chromatographic separation was achieved on a C(18) column using gradient elution in a water-acetonitrile system containing 0.1% (v/v) formic acid. The tandem mass spectrometric analysis was performed in the multiple reaction monitoring mode using internal standardization to improve assay precision and accuracy. For plasma sample pretreatment, acetonitrile was added first to precipitate proteins and SPE was used to minimize matrix effects. Using LC-ESI-MS-MS, the LOQ was 0.5 ng ml(-1) for DADLE and 2 to 5 ng ml(-1) for its prodrugs. Good linearity was observed from the LOQ up to 1000 ng ml(-1) for all compounds. For the analysis of DADLE, both analytical methods showed good precision, accuracy and stability. However, for prodrug analysis, LC-FLU showed some sensitivity and accuracy problems, while the LC-ESI-MS-MS method provided consistent and satisfactory results. In conclusion, LC-ESI-MS-MS is the method of choice for the analysis of DADLE and its cyclic prodrugs in rat plasma samples due to its good selectivity, high sensitivity, and fast analysis. Its application was demonstrated through biodisposition and bioconversion studies of the coumarinic acid-based prodrug after intravenous administration in rats.

Transport and metabolism of opioid peptides across BeWo cells, an in vitro model of the placental barrier.

In keeping with the advance of biotechnology, cell culture becomes an important tool for investigating the transport and the metabolism phenomena. A cell line of human origin, the BeWo choriocarcinoma cell line, was used for the study of the transport and metabolism of opioid peptides across the in vitro model of the placental barrier. Opioid peptides, both naturally occurring and their synthetic analogs, are of interest to be developed as potent analgesics and were included in this study. The apparent permeability coefficients (Pe)s of the peptides containing 4-11 amino acid or analog residues were in the range of 0.23-14.6 x 10(-5) cm/s. The (Pe)s of these peptides were comparable to those of sucrose or dextrans, hydrophilic markers. The (Pe)s of low molecular weight (MW) peptides was not dependent on their MW or molecular size, whereas an inversely linear correlation between (Pe)s and molecular size was observed with the larger peptides. Molecular sieving of the BeWo monolayer restricted the transport of the peptides with MW> or =1033 Da or molecular size > or =6.6 A. Membrane partitioning ability and charge of the peptides were also investigated and found to be the minor factors regulating the extent of peptide permeation. Contrasting to the transport of Tyr-[D-pen-Gly-Phe-D-Pen] (DPDPE) peptide analog across the blood-brain barrier, the transport of DPDPE across the BeWo monolayers were not indicated to be via carrier-mediated transport. The major transport pathway of the opioid peptides across the BeWo monolayers was found to be via paracellular route. In metabolism studies, aminopeptidase was found to be a major enzyme type responsible for the degradation of naturally occurring peptides but not for the synthetic analogs. The finding obtained from the present study reveals the applicability of the BeWo cell line as an in vitro model for investigating placental transport and metabolism of opioid peptides.

Target specific sample preparation from aqueous extracts with molecular imprinted polymers.

In this paper we report a method for the synthesis of molecular imprinted polymers for use in sample preparation with aqueous biological materials. Highly cross-linked bulk polymers were synthesized in the presence of the template molecule, 2,6-pyridinedicarboxylic acid (DPA) using acrylamide (ACD) and 4-vinylpyridine (VP) as functional monomers. Conditions are described for the optimization of the template complex with temperature, copolymer mixture and crosslinker type. Selective binding of the template molecule is demonstrated in comparison to structural isomers and analogs for molecular imprinted polymers (MIPs) synthesized with three different crosslinkers, ethyleneglycol dimethacrylate (EGDMA), bisacrylamide and N,N'-1,3-phenylene bismethacrylamide (PBMA). The chromatographic capacity factors and selectivities for a series of structural analogs were compared. Molecular imprinted polymers prepared with equimolar ratios of ACD and VP and either PBMA or bisacrylamide resulted in highly selective binding for the template versus analogs with similar structure and chemistry. Multiple molecular dissociation constants were measured with the maximum binding capacities for EGDMA, PBMA and bisacrylamide measuring 17, 27 and 90 micromol/g, respectively.