Evaluation and optimization of capillary zone electrophoresis with different dynamic capillary coatings for the determination of carbohydrate-deficient transferrin in human serum

Serum transferrin (Tf) comprises several isoforms with up to two complex oligosaccharide chains containing zero to eight sialic acid residues and neutral sugars. The major glycoform, known as tetrasialo-Tf, contains four sialic acid residues and accounts for about 80% of whole Tf in human serum. Carbohydrate-deficient transferrin (CDT) encompasses isoforms that are deficient in carbohydrate chains and consequently in sialic acid residues (including asialo-, monosialo- and disialo-Tf) and is a well known marker for chronic alcohol abuse. Recently capillary zone electrophoresis (CZE) has been reported as a tool extremely effective for the simultaneous, individual, quantitative determination of CDT isoforms. Three CZE methods that feature different dynamic capillary coatings were evaluated and optimized for CDT determination in human serum of alcohol abusers and control subjects. CZE separation was performed in alkaline borate buffers after serum sample saturation with iron, electropherograms were detected at 200 nm, data were evaluated as % area of disialo-Tf in relation to tetrasialo-Tf and peak identification was accomplished via relative migration times to tetrasialo-Tf, immunosubtraction and enzymatic sequential cleavage of sialic acid residues. Dynamic capillary coatings with diaminobutane, spermine and a double coating produced by commercially available proprietary agents were investigated and found to be suitable for determination of CDT in human serum. For all three approaches, best results were obtained in 50 microm I.D. fused-silica capillaries of 50 cm effective length and a capillary cartridge temperature of 20-25 degrees C. Using 3 mM 1,4-diaminobutane or 0.02 mM spermine in a borate-based running buffer of pH 8.3 provided data of remarkable similarity with resolution of di-, tri-, tetra- and pentasialo-Tf within 15-18 min. With the double coating, asialo-Tf and Tf isoforms with two to six sialic acid residues were baseline separated. Compared to the two amine-based procedures, the run times were found to be somewhat shorter, the detector signals higher, the applied power level significantly lower and the reproducibility better.     

Capillary zone electrophoresis with a dynamic double coating for analysis of carbohydrate-deficient transferrin in human serum. Precision performance and pattern recognition

Capillary zone electrophoresis (CZE) with a dynamic double coating permits the simultaneous, individual, quantitative determination of transferrin (Tf) isoforms in human serum and thus carbohydrate-deficient transferrin (CDT), the most specific marker available today for the detection of chronic, excessive alcohol intake. CZE of serum Tf was carefully evaluated using the P/ACE MDQ with fused-silica capillaries of 50 microm I.D. and 60.2 cm total length, the CEofix CDT kit and the instrumental conditions recommended by the kit manufacturer. The precision performance assessed over a 20-day period according to the internationally accepted NCCLS EP5-A guidelines revealed the CZE assay as being highly reproducible with within-run and total precision being dependent on the Tf isoform level and RSD values ranging between 2.2 and 17.6%. Inter-day RSD values for asialo-Tf were noted to be between 9.8 and 11.5% and for disialo-Tf between 3.8 and 8.6%, whereas those for CDT levels of 0.87 and 4.31% of total Tf were determined to be 8.6 and 3.4%, respectively. The RSD values for trisialo-Tf, tetrasialo-Tf, pentasialo-Tf and hexasialo-Tf were found to be between 0.4 and 4.1%. Tf patterns are recognized and identified via detection times of Tf isoforms (intra-day and inter-day RSD values < 1.0% and < 1.7%, respectively), immunosubtraction of Tf and enzymatic sequential cleavage of sialic acid residues. Furthermore, heterozygous Tf BC and Tf CD variants are assigned via spiking with a known mixture of Tf isoforms (e.g. the serum of a healthy Tf C homozygote). Among the non-Tf peaks monitored, the CRP peak detected shortly before disialo-Tf was identified by immunosubtraction and peak magnitudes were found to correlate well with immunochemically determined CRP serum levels. The CZE assay with dynamic double coating could thereby be shown to be sensitive enough to determine elevated CRP levels in human serum. Furthermore, unusual peaks in the gamma-region were identified by customary serum protein CZE, immunosubtraction CZE and immunofixation.     

Electrospray ionization mass spectra of hemoglobin and transferrin by a magnetic sector mass spectrometer. Comparison with theoretical isotopic distributions.

Electrospray ionization mass spectra of human hemoglobin chains and of transferrin were acquired on a magnetic sector mass spectrometer. The observed molecular ion for each hemoglobin chain was in good agreement with the theoretical isotopic distribution at a reasonable resolution of 2000. The clear separation of a variant beta-chain in admixture with the normal counterpart at mass 15,867 that differed from it by 14 Da (0.09%) ensured that a smaller mass difference could be detected. The molecular ions for human transferrin were too broad compared with the theoretical shape to determine the molecular mass accurately, probably due to the heterogeneity of the carbohydrate moiety. A decrease in mass by neuraminidase digestion, however, determined the average number of sialic acids in the molecule.     

Transferrin immunoextraction for determination of carbohydrate‐deficient transferrin in human serum by capillary zone electrophoresis

CZE‐based assays for carbohydrate‐deficient transferrin (CDT) in which serum is mixed with an Fe(III) ion‐containing solution prior to analysis are effective approaches for the determination of CDT in patient samples. Sera of patients with progressed diseases, however, are prone to interferences comigrating with transferrin (Tf) that prevent the proper determination of CDT by CZE in these samples. The need of a simple and economic approach to immunoextract Tf from human serum prompted us to investigate the use of a laboratory‐made anti‐Tf spin column containing polyclonal rabbit anti‐human Tf antibodies linked to Sepharose 4 Fast Flow beads. This article reports extraction column manufacturing and column characterization with sera having normal and elevated CDT levels. The developed procedure was applied to a number of relevant hepatology and dialysis patient samples and could thereby be shown to represent an effective method for extraction and concentration of all Tf isoforms. Furthermore, lipemic sera were delipidated using a mixture of diisopropyl ether and butanol prior to immunoextraction. CDT could unambiguously be determined in all pretreated samples.     

DBAH-Tf靶向药物及其对乳腺癌细胞的杀伤作用分析

以转铁蛋白(Tf)为靶向载体,与双氢青蒿素(DHA)化学偶联制备一种新的双氢青蒿素衍生物-转铁蛋白靶向药物.以DHA为原料先合成了12β-对甲酰肼苯基双氢青蒿素(DBAH),并采用紫外、红外、核磁共振及电化学等手段对目标产物的结构进行了表征.利用高碘酸钠氧化Tf的C端的N-糖链上的邻位羟基,其氧化产物和DBAH通过希夫碱偶联合成了DBAH-Tf靶向药物,采用紫外吸收光谱法和电化学方法进行表征.四甲基氮唑蓝法(MTT)分析了DBAH-Tf及DHA对人乳腺癌细胞(MCF-7)和正常乳腺细胞的体外杀伤作用, 结果表明,DBAH-Tf对MCF-7的杀伤作用是正常乳腺癌细胞的286倍,体现出良好的靶向性.     

Molecularly Imprinted Polymer Coated Quantum Dots for Multiplexed Cell Targeting and Imaging

Advanced tools for cell imaging are of great interest for the detection, localization, and quantification of molecular biomarkers of cancer or infection. We describe a novel photopolymerization method to coat quantum dots (QDs) with polymer shells, in particular, molecularly imprinted polymers (MIPs), by using the visible light emitted from QDs excited by UV light. Fluorescent core–shell particles specifically recognizing glucuronic acid (GlcA) or N‐acetylneuraminic acid (NANA) were prepared. Simultaneous multiplexed labeling of human keratinocytes with green QDs conjugated with MIP‐GlcA and red QDs conjugated with MIP‐NANA was demonstrated by fluorescence imaging. The specificity of binding was verified with a non‐imprinted control polymer and by enzymatic cleavage of the terminal GlcA and NANA moieties. The coating strategy is potentially a generic method for the functionalization of QDs to address a much wider range of biocompatibility and biorecognition issues.     

Exact molecular mass determination of various forms of native and de-N-glycosylated human plasma-derived antithrombin by means of electrospray ionization ion trap mass spectrometry

Human plasma-derived antithrombin was characterized in both the native and de-N-glycosylated forms (without separation of isoforms) by means of electrospray ionization ion trap mass spectrometry (ESI-ITMS). In order to determine the limits of the instrument set-up, the molecular mass precision and accuracy of the ESI-ITMS analysis was evaluated with the standard protein enolase and some instrumental data acquisition parameters were optimized. Mass precision was determined as a function of the number of averaged mass spectra (= scans) and data acquisition time. For this study, 20 and 50 scans were averaged and the data acquisition time was chosen to be between 0.5 and 5 min. It turned out that data acquisition times longer than approximately 2 min show no significant differences of the standard deviation of the determined molecular mass. Furthermore, the ion trap scan rate was varied at constant acquisition time of 2 min and the number of averaged scans was set to 20. At the scan rate of 13,000 u s(-1) a mass precision of +/-1.8 Da and a mass accuracy of +0.026% were determined. On reducing the scan rate to 5500 u s(-1), better agreement with the theoretical molecular mass was obtained, showing a mass accuracy of +0.012% but with a decrease in the mass precision to +/-3.0 Da. Using the optimized scan rate of 13,000 u s(-1) and a data acquisition time of 2 min, the exact molecular mass was determined of the three forms of antithrombin, namely the alpha-form, the beta-form and the natural mixture (present in human plasma) containing both forms. The protonated molecular masses were found to be 57,854 and 55,664 Da for the affinity chromatography-isolated alpha-and beta-form, respectively. The mass difference of 2190 Da is attributed to the known difference in carbohydrate content at one specific site. The protonated molecular mass of the dominating species of the natural mixture in human plasma was shown to be 57,850 Da, corresponding to the alpha-form, the major component in native plasma. In this mixture the beta-form was also detected, exhibiting a protonated molecular mass of 55,655 Da, but showing a much lower abundance, as expected. To obtain a complete release of the N-glycan residues by means of PNGase F, a denaturation, reduction and alkylation step of the glycoproteins was performed before the enzymatic reaction. After enzymatic removal of all N-glycans, the protonated molecular masses obtained were 49,399, 49,380 and 49,391 Da for the alpha-form, the beta-form and the unseparated natural mixture, respectively. These values are in good agreement (+0.026% for the alpha-form, -0.012% for the beta-form and +0.010% for the unseparated mixture) with the calculated molecular mass based on the SwissProt data. The determined molecular masses after reduction/alkylation and de-N-glycosylation of the alpha-and beta-forms are almost equal, indicating that no major differences exist between the three preparations on the amino acid level.     

Purification of transferrin by magnetic immunoaffinity beads

Immunoaffinity adsorbent for transferrin (Tf) purification was prepared by immobilizing anti‐transferrin (Anti‐Tf) antibody on magnetic monosizepoly(glycidyl methacrylate) beads, which were synthesized by dispersion polymerization technique in the presence of Fe₃O₄nanopowder and obtained with an average size of 2.0 μm. The magnetic poly(glycidyl methacrylate) (mPGMA) beads were characterized by Fourier transform infrared spectroscopy, swelling tests, scanning electron microscopy, electron spin resonance spectroscopy, thermogravimetric analysis and zeta sizing analysis. The density and swelling ratio of the beads were 1.08 g/cm³ and 52%, respectively. Anti‐Tf molecules were covalently coupled through epoxy groups of mPGMA. Optimum binding of anti‐Tf was 2.0 mg/g. Optimum Tf binding from aqueous Tf solutions was determined as 1.65 mg/g at pH 6.0 and initial Tf concentration of 1.0 mg/mL. There was no remarkable loss in the Tf adsorption capacity of immunoaffinity beads after five adsorption–desorption cycles. Tf adsorption from artificial plasma was also investigated and the purity of the Tf molecules was shown with gel electrophoresis studies.     

Capillary zone electrophoresis for determination of carbohydrate-deficient transferrin in human serum

Carbohydrate-deficient transferrin (CDT) is the most specific marker for diagnosis of chronic excessive alcohol consumption and includes the serum transferrin (Tf) isoforms with two or less sialic acid residues (di-, mono-, and asialo-Tf). To monitor serum CDT, we developed a capillary zone electrophoresis (CZE) method based on the dynamic capillary coating with diethylenetriamine (DETA). The separation was performed in a bare fused-silica capillary (50 microm ID, 57 cm in length), applying a voltage of 25 kV and a temperature of 40 degrees C. Using a 100 mmol/L borate buffer, pH 8.4 with 3 mmol/L DETA, the Tf isoforms (asialo- to pentasialo-Tf) were resolved within 16 min. Enzymatic cleavage of sialic acid residues with neuraminidase and immunosubtraction were used to identify CDT isoforms. The relative amount of CDT expressed as area % of disialo-Tf isoform related to the area of tetrasialo-Tf in 50 healthy donors (24 males and 26 females; aged 25-50 years) was 3.15 +/- 0.76% (mean +/- SD). The comparison between CDT values obtained by this CZE procedure and the "Axis-Shield %CDT" kit gave r = 0.644, p < 0.001 (n = 290). This easy to use and inexpensive CZE procedure could be an ideal tool to investigate CDT proteins for clinical or forensic purposes.     

Primary structure of β-momorcharin,a ribosome-inactivating protein from the seeds of Momordica Charantia Linn (Cucurbitaceae)

The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S.aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 51 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.     

Common Pathway for K562 Cells Endocytosis and Release of Gac-Tf and Ga2-Tf via a Transferrin Receptor

There has been an increasing interest in the use of gallium in anticancer activity. However, whether the uptakes of two species of transferrin, including digallium transferrin (Ga₂‐Tf) and the C‐terminal monogallium transferrin (Ga_C‐Tf) by cells, are different is not well understood. In this work the mechanism of both species passing in and out K562 cells has been established by using ¹²⁵I‐labeled transferrin. There were about (1.5±0.08)×10⁵ binding sites per cell surface. Both Ga₂‐Tf and Ga_C‐Tf were recycled to the cell exterior with a protracted endocytic cycle compared to apotransferrin (apoTf). The cycling time from the internalization to release was calculated about t_1/2= (3.15±0.055) min for apoTf, t_1/2= (4.69±0.09) min for Ga₂‐Tf and t_1/2= (4.78±0.15) min for Ga_C‐Tf. The result implies that metal dissociating from transferrin in acidic endosomes was likely to be the key step. Both Ga₂‐Tf and Ga_C‐Tf into K562 cells are transferrin receptor‐mediated process with a similar rate of endocytosis and release. Our present observations provide useful information for better targeted drugs in specific therapy.     

Estimation of sialic acid in a sialoglycan and a sialoglycoprotein by capillary electrophoresis with in-capillary sialidase digestion

An example of application of in-capillary derivatization for CE, obtained by using the throughout-capillary format, is presented. Introduction of a sialoglycan (N-acetylneuraminyllactose) or a sialoglycoprotein (bovine serum fetuin) sample to a running buffer (pH 5.0) containing N-acetylneuraminidase followed by application of a voltage resulted in the release of N-acetylneuraminic acid (NANA) which could be estimated by CE with UV detection. Two-step application of voltages (5 and 20 kV) was proved to be more effective for rapid estimation of the released NANA. This format (modified throughout-capillary format) allowed differential estimation of the NANA present in the sample as an impurity and the NANA released from the substrate at the picomol level, and thereby reliable micro assay of the sialidase activity. It also allowed estimation of the rate constant of this enzymatic reaction.     

The effect of covalent immobilization of sialic acid on the removal of lipopolysaccharide and reactive oxygen species for polyethylene terephthalate

Polyethylene terephthalate (PET) was aminolyzed with 1,6‐diaminohexane (DAH) and then sialic acid (NANA) was immobilized via amidation onto the surface. The surface concentration of NANA was determined by 2‐thiobarbituric acid (TBA) test. The hemocompatibility of the resulting PET fabrics was evaluated based on complete blood count (CBC), coagulating times, and protein adsorption. The ability to remove lipopolysaccharide (LPS) was also determined. In addition, the effect of contacting NANA‐immobilizing PET on the suppression of reactive oxygen species (ROS) production was measured by the chemiluminescence (CL) method. The results show that by immobilizing NANA onto PET, the adhesion of platelet (PLt) was reduced, and oxidative stress was suppressed. The level of LPS was also greatly reduced. Copyright © 2010 John Wiley & Sons, Ltd.     

Improved sensitivity for insulin in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by premixing alpha-cyano-4-hydroxycinnamic acid matrix with transferrin

This report describes an enhancement of the signal intensities of proteins and peptides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). When alpha-cyano-4-hydroxycinnamic acid (CHCA) premixed with human transferrin (Tf) was used as a matrix, the signal intensity of insulin was amplified to more than ten times that of the respective control in CHCA without Tf. The detection limit of insulin was 0.39 fmol on-probe in the presence of Tf, while it was 6.3 fmol in the absence of Tf. The signal intensity of insulin was also enhanced when the CHCA matrix was premixed with proteins other than Tf (80 kDa), such as horse ferritin (20 kDa), bovine serum albumin (BSA, 66 kDa), or human immunoglobulin G (150 kDa). The optimum spectrum of insulin was obtained when the added amount of protein was in the range 0.26-0.62 pmol, regardless of the molecular weight of the added protein. Tf and BSA outperformed the other tested proteins, as determined by improvements in the resulting spectra. When the mass spectra of several peptides and proteins were recorded in the presence of Tf or BSA, the signal intensities of large peptides such as glucagon were enhanced, though those of smaller peptides were not enhanced. In addition, the signal enhancement achieved with Tf and BSA was more pronounced for the proteins, including cytochrome C, than for the large peptides. This enhancement effect could be applied to improve the sensitivity of MALDI-TOFMS to large peptides and proteins.     

Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins

The human plasma protein patterns obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a good model system for post-translational modifications because of the existence of several "ladders" of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883-906], so-called "trains" of spots. Our investigation of several proteins, amongst others beta2-microglobulin and the haptoglobin chains, found the differences in isoelectric points (p/) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2-D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separated and quantified by reversed-phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine-containing and deamidated asparagine-containing peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2-D gel consists of a mixture of protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the "ladder" is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.     

A mid-IR flow-through sensor for direct monitoring of enzyme catalysed reactions. Case study: Measurement of carbohydrates in beer

A novel mid-IR flow-through sensor for in situ monitoring of the enzymatic reaction of amyloglucosidase with carbohydrates was developed. Amyloglucosidase was immobilised on agarose beads with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and directly placed in a conventional IR flow-through cell. The carbohydrate content of various beer samples was then determined by following the enzymatic hydrolytic cleavage of carbohydrates to glucose with Fourier-transform infrared (FTIR) spectroscopy. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating the flow-through sensor in a sequential injection (SI) manifold. As the immobilised enzyme was directly probed by the IR beam, an in situ study of the enzymatic reaction was possible enabling determination of the Michaelis-Menten constant of the immobilised enzyme. A linear calibration curve was recorded using maltose standards in the range between 0.86 and 7.13 g L(-1). The proposed method was successfully applied to the determination of the carbohydrate content of four different beer samples by the standard addition method. Moreover experiments showed the possibility of monitoring in situ the immobilisation of the enzyme as well as a small organic acid (malic acid) on the agarose beads using EDC.     

钴酞菁功能化聚合物整体柱的制备及用于转铁蛋白糖肽的富集

建立了一种新型的聚合物整体柱制备方法,将四羧基钴酞菁（CoPcTc）引入聚（甲基丙烯酸缩水甘油酯-乙二醇二甲基丙烯酸酯）（Poly（GMA-EDMA））整体柱,用于转铁蛋白糖肽的富集。由于四羧基钴酞菁和糖肽之间的氢键和金属配位作用,制备的整体材料在用于糖肽富集时具有高效性和选择性。转铁蛋白经过功能化整体柱富集后,通过电喷雾四极杆飞行时间质谱分析,共捕获到17条糖肽。当转铁蛋白浓度降至8.8×10^-10mol/L时,仍然可以得到3条糖肽,说明合成的聚合物整体材料对微量蛋白样品分析具有很高的应用潜力。     

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction--under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF--using infiltration-centrifugation procedure--that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.     

Metabolite identification of arbidol in human urine by the study of CID fragmentation pathways using HPLC coupled with ion trap mass spectrometry

The metabolism of arbidol in humans was studied using liquid chromatography-electrospray ionization (ESI) ion trap mass spectrometry (ITMS) after an oral dose of 300-mg arbidol. A total of 17 metabolites were identified including the glucuronide arbidol and the glucuronide sulfinylarbidol as the major metabolites.Arbidol and its metabolites have some common fragmentation patterns as a result of a homolytic bond cleavage. This cleavage will form odd-electron ions with the loss of a radical. The arbidol fragmentation sequence is first to lose dimethylamine (45 Da), followed by the loss of acetaldehyde (44 Da), and then the phenylthio radical (109 Da). This fragmentation sequence is also observed from N-demethylarbidol, sulfonylarbidol, and N-demethylsulfonylarbidol. However, for sulfinylarbidol and N-demethylsulfinylarbidol, the fragmentation sequence is reversed so that the phenylsulfiny radical (125 Da) was lost first, followed by the loss of dimethylamine (45 Da), and then acetaldehyde (44 Da). The exact masses for arbidol and sulfinylarbidol fragment ions were determined by a quadrupole/time-of-flight mass spectrometer (Q-TOF MS).The phase II metabolites, such as sulfate and glucuronide conjugates of arbidol, N-demethylarbidol, sulfonylarbidol, and N-demethylsulfonylarbidol were identified by observing the neutral loss of 80 Da (SO(3)) or 176 Da (glucuronic acid) from the MS(2) spectra. The sulfate and glucuronide conjugates such as sulfinylarbidol and N-demethylsulfinylarbidol had an unusual fragmentation pattern, in which the phenylsulfinyl radical (125 Da) was lost before the loss of SO(3) group (80 Da) or glucuronic acid (176 Da) occurred.     

Electrospray mass spectrometry for the direct accurate mass measurement of ligands in complex with the retinoid X receptor α ligand binding domain

Accurate mass measurements are often used in the structural determination of unknown compounds of low molecular mass (i.e., below approximately 500 Da). Recently, it has been shown that accurate mass measurements also can be made on small denatured proteins (i.e., M(r), approximately 17,000) to confirm their amino acid composition and identify the presence of isoforms. In the current report, we present nondenaturing electrospray (ES) mass spectrometry data on the direct accurate mass measurement of ligands in complex with the retinoid X receptor ligand binding domain (RXR LBD; M(r) 31,370.92). Average mass errors were below 0.198 Da, 6.3 ppm (standard deviation [SD], 0.146; n = 10) for low-affinity fatty acid agonists analyzed in complex with the RXR LBD. Protein consumption was less than 15 pmol, with fatty acid ligands present at concentrations corresponding to their median effective concentration value (low micromolar, determined in transfection assays). Although determination of fatty acid mass was only sufficiently accurate to give nominal mass values, measurements were of sufficient accuracy to assign fatty acid chain length, degree of unsaturation, or cyclization. Using 17beta-estradiol as a control, the ability to observe specific ligand binding is shown for both high- and low-affinity RXRalpha agonists. In addition, binding of a novel synthetic receptor agonist XCT0315908 to the RXRalpha LBD is reported. This compound showed a high degree of complex formation, and the receptor-ligand complex could be mass measured with an average mass error of -0.024 Da, 0.8 ppm (SD, 0.092; n = 9). Thus, specific binding of both nanomolar and micromolar affinity ligands to a nuclear receptor LBD can be directly observed using nondenaturing ES mass spectrometry and accurate mass measurements additionally can be made on intact complexes in the same experiment. This methodology also is applicable when ligands are present as components of mixtures.