HPLC-electrospray Mass Spectrometry Research Articles

Convergence of Three Methods to Resolve Discrepant Immunoassay Digitoxin Results

We have observed significant analytical discrepancies (20% to 220%) in digitoxin (Crystodigin; DTN) immunoassay results for 11 serum samples from patients taking digitoxin. We used three methods—HPLC, immunoassay, and liquid chromatography electrospray mass spectrometry (LC/MS)—to investigate the possible sources of these discrepancies. DTN is indicated for treatment of heart failure, atrial flutter, and supraventricular tachycardia (1). This drug is the most slowly excreted cardiac glycoside, with a half-life of 4 to 10 days (2). Administration and therapeutic monitoring of DTN are more popular in European countries than in the US. Plasma concentrations of 15–25 μg/L are considered therapeutic (3), and values >35 μg/L have been associated with toxicity in 80% of patients (4). Biotransformation of DTN is complex. It is oxidatively cleaved to bisdigitoxoside (bis-DTN) in the liver, catalyzed by a specific cytochrome P-450 enzyme (5). Further deglycosylation to monodigitoxoside (mono-DTN) and digitoxigenin has been documented in mice, rabbits, and guinea pigs (5). DTN can be hydroxylated at position 12 to form digoxin. Glucuronidation of mono-DTN and, to a lesser extent, of digitoxigenin to form polar conjugates has also been documented (6). In isolated guinea pig atria, the positive inotropic actions of DTN and its metabolites have been ranked in order of decreasing activity as follows: mono-DTN, bis-DTN, digoxin, DTN, digitoxigenin (7). Discrepant patients’ samples had been analyzed by the following immunoassays: ACS:180® (Chiron Diagnostics), which utilizes a monoclonal antibody with chemiluminescence detection; TDx® (Abbott Labs.), which utilizes a polyclonal antibody and fluorescence polarization detection; and DPC (Diagnostic Products Corp.), which uses a polyclonal antibody with RIA technology. Patients’ samples had been collected in Germany in accordance with the institutional human studies regulations of that country. All immunoassays had been performed according to their manufacturers’ instructions. Initially, we expected the deglycosylated congeners of …

Determination of naphthodianthrones in plant extracts from Hypericum perforatum L. by liquid chromatography–electrospray mass spectrometry

The determination of the naphthodianthrone constituents in extracts of dried blossoms of Hypericum perforatum L. by combined HPLC–electrospray mass spectrometry is described. Hypericin (1), pseudohypericin (2) and their precursor compounds produce intensive negative quasi-molecular ions by deprotonation provided a non-acidic eluent system is used in the HPLC separation. From the [M–H] − ions formed in the electrospray ionization process characteristic daughter ion spectra can be obtained by collisional activation which have been studied by tandem mass spectrometry.

Determination of glucosinolates in domestic and wild mustard by high-performance liquid chromatography with confirmation by electrospray mass spectrometry and photodiode-array detection

A method has been developed to easily separate and quantify glucosinolates in mustard and winter cress plants using reversed-phase HPLC with volatile ion-pairing reagents to modify the mobile phase and enhance retention and selectivity. Volatile buffers are required for HPLC-electrospray mass spectrometry. Confirmation of identity was accomplished using both negative ion electrospray mass spectrometry and photodiode-array detection. This method provides a way to determine glucosinolates in Brassica and cruciferous vegetables, most of which are commercially unavailable. Glucosinolates were determined in various parts and sections of yellow and brown domestic mustards and winter cress (wild mustard). Results varied from 0.2 mg/g glucosinolate content in the roots of the plants to 26 mg/g in the seeds.

On-line HPLC electrospray mass spectrometry of phosphorothioate oligonucleotide metabolites.

Metabolism of 2'-deoxyphosphorothioate oligonucleotides ISIS 11061 and ISIS 11637 was examined with capillary gel electrophoresis (CGE) and on-line HPLC electrospray mass spectrometry (HPLC/ES-MS). Oligonucleotides were isolated from plasma, liver, and kidneys of rats injected with ISIS 11061 and ISIS 11637. Metabolites found in plasma were consistent with 3'-exonuclease activity. Metabolites isolated from liver and kidney were consistent with 3'- and/or 5'-exonuclease activity. HPLC/ES-MS analysis of ISIS 11061 isolated from kidney indicated extensive degradation from the 3' terminus, but metabolites consistent with 5' degradation and combinations of 3' and 5' truncations also were observed. ISIS 11061 isolated from liver showed less extensive degradation. The 5' truncated metabolites represented the predominant species in contrast to the kidney sample. Metabolites with masses consistent with combinations of 3' and 5' truncations were also observed in liver. The metabolic profiles generated by CGE analysis of these samples agreed qualitatively with mass spectrometric results. HPLC/ES-MS enabled the simultaneous determination of degradation products that are the same length but differ in composition. CGE could discriminate species that differed by one nucleotide in length. HPLC/ES-MS was shown to be a useful tool to study the complex metabolism of antisense oligonucleotides in vivo.

Separation and characterization of the metal-thiolate-cluster domains of recombinant sea urchin metallothionein.

Partial metal depletion and 113Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrotus purpuratus (Cd7-MTA) binds its metal ions in a four-metal (Cd4Cys11) and a three-metal (Cd3Cys9) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E.A., Zerbe, O., Hess, D., Hunziker, P.E., Vasak, M. & Kägi, J.H.R. (1995) Biochemistry 34,7460-7467]. This partitioning has now been confirmed by bisecting native Cd7-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37-39 residues with four Cd ions and a set of C-terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal-bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N-terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7-MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster.

Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring.

HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within "high-probability" PKA consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three criteria were used to assign phosphorylated sites: (1) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; (2) co-elution with the PO3- ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The "low-probability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.

Post‐Translational Heterocyclic Backbone Modifications in the 43‐Peptide Antibiotic Microcin B17

Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nature, is produced by ribosomal synthesis and post-translational modification of the 69-residue precursor protein by an Escherichia coli strain. To elucidate the chemical structure of the mature 43-residue peptide antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substantial amounts of highly pure McB17 (non-labelled, 15N-labelled and 13C/15N-labelled peptide. By ultraviolet-absorption spectroscopy. HPLC-electrospray mass spectrometry and GC-mass spectrometry, amino acid analysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modification of the precursor backbone at Gly-Cys and Gly-Ser segments leads to the formation of 2-aminomethylthiazole-4-carboxylic acid and 2-aminomethyloxazole-4-carboxylic acid, respectively. In addition, two bicyclic modifications 2-(2-aminomethyloxazolyl)thiazole-4-carboxylic acid and 2-(2-aminomethylthiazolyl)oxazole-4-carboxylic acid were found that consist of directly linked thiazole and oxazole rings derived from one Gly-Ser-Cys and one Gly-Cys-Ser segment. Analogous to the thiazole and oxazole rings found in antitumor peptides of microbial and marine origin, these heteroaromatic ring systems of McB17 presumably play an important role in its gyrase-inhibiting activity, e.g. interacting with the DNA to trap the covalent protein-DNA intermediate of the breakage-reunion reaction of the gyrase.

S-oxidative cleavage of farnesylcysteine and farnesylcysteine methyl ester by the flavin-containing monooxygenase.

Posttranslational modification of proteins with a farnesyl or geranylgeranyl group appears to be crucial in the signal transduction of eukaryotic cells. For example, farnesylation of ras-encoded proteins is a key process that apparently leads to membrane association of proteins that perform a function in cell growth-promoting activity. Although it has been suggested that prenylation of proteins may be an important regulatory mechanism, little is known about the mechanism whereby prenylated proteins are removed from the membrane. In our previous report [(1992) Chem. Res. Toxicol. 5, 193-201], we showed that S-alkenylated cysteines and mercapturates of xenobiotics were S-oxygenated by the flavin-containing monooxygenase. The S-oxides were not indefinitely stable and rearranged or underwent elimination reactions that cleaved the C-S(O) bond. As a model for farnesylated proteins and peptides, the biotransformation of farnesylcysteine methyl ester was examined in the presence of pig liver microsomes. Two prominent products were formed: farnesyl methyl ester sulfoxide and farnesylcysteine, arising from the action of the flavin-containing monooxygenase and esterase of pig liver, respectively. Formation of farnesylcysteine methyl ester sulfoxide by the flavin-containing monooxygenase was stereoselective (i.e., 71.5%:28.5%, major to minor diastereomer) in good agreement with previously reported stereoselectivity studies of other related S-alkylcysteine-containing compounds. That the stereoselectivity observed was due to S-oxygenation of the sulfur atom was verified in parallel chemical oxidation studies by using micellar electrokinetic capillary chromatography. Farnesylcysteine methyl ester was an excellent substrate for the flavin-containing monooxygenase, and the S-oxide product was confirmed by HPLC electrospray mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Ca2+-Binding S100 Proteins in Human Heart by HPLC-Electrospray Mass Spectrometry

Calcium dependent processes are often impaired in myocardial dysfunctions and calcium-binding proteins might be involved as mediators of Ca2+ signals. Here we present a method to assess a footprint of the calcium-binding proteins of the S10O family in human heart. The S100 proteins are purified through calcium dependent affinity chromatography and reverse phase high-performance liquid chromatography and are analyzed by electrospray-ionization mass spectrometry (ESI-MS) and liquid secondary ionization/tandem quadrupole mass spectrometry (LS-MS/MS). In human heart we identified S100α, CACY, and CAPL as the most abundant S100 proteins and showed that they occur as monomers and homodimers.