Targeted ion parking for the quantitation of biotherapeutic proteins: concepts and preliminary data.

Targeted ion parking (or TIPing) is the first quantitative application of ion/ion reactions for mass spectrometry. In TIPing, intact biotherapeutic proteins are electrosprayed as intact molecules (no digestion) and, as expected, many multiply protonated species are produced (e.g., (M + 7H)(7+), (M + 8H)(8+), etc.). Several of these multiply charged species are selectively isolated using a quadrupole mass analyzer and then contained in a linear ion trap. The protein ions are then subjected to a proton-transfer reaction with a reagent anion. The ions undergo sequential charge reduction (e.g., to (M + 6H)(6+)) during a defined reaction period. Applying a low-amplitude waveform to the trap during this reaction time stops the ion/ion reaction at a chosen (and predicted) charge state for the protein. This funnels the analyte ions into a single channel with relatively high efficiency (>50% of reactant ion signal is converted into product ion signal) that can be used for quantitation. In TIPing, the target protein's molecular weight and charge state distribution are the only prerequisite knowledge required. This information can be acquired experimentally or can be easily predicted based upon amino acid sequences. Preliminary data for a biotherapeutic protein, a domain antibody, were collected using TIPing coupled online with liquid chromatography (LC-TIPing). The LC-TIPing data demonstrate a linear response for samples from 10-1000 ng/mL extracted from a complex plasma sample, demonstrating the analytical potential for TIPing.

Rapid sensitive analysis of cysteine rich peptide venom components.

Disulfide-rich peptide venoms from animals such as snakes, spiders, scorpions, and certain marine snails represent one of nature's great diversity libraries of bioactive molecules. The various species of marine cone shells have alone been estimated to produce >50,000 distinct peptide venoms. These peptides have stimulated considerable interest because of their ability to potently alter the function of specific ion channels. To date, only a small fraction of this immense resource has been characterized because of the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and can contain extensive posttranslational modifications. The extraordinary complexity of crude venoms and the lack of DNA databases for many of the organisms of interest present major analytical challenges. Here, we describe a strategy that uses mass spectrometry for the elucidation of the mature peptide toxin components of crude venom samples. Key to this strategy is our use of electron transfer dissociation (ETD), a mass spectrometric fragmentation technique that can produce sequence information across the entire peptide backbone. However, because ETD only yields comprehensive sequence coverage when the charge state of the precursor peptide ion is sufficiently high and the m/z ratio is low, we combined ETD with a targeted chemical derivatization strategy to increase the charge state of cysteine-containing peptide toxins. Using this strategy, we obtained full sequences for 31 peptide toxins, using just 7% of the crude venom from the venom gland of a single cone snail (Conus textile).

High sensitivity 1H-NMR spectroscopy of homeopathic remedies made in water.

BACKGROUND: The efficacy of homeopathy is controversial. Homeopathic remedies are made via iterated shaking and dilution, in ethanol or in water, from a starting substance. Remedies of potency 12 C or higher are ultra-dilute (UD), i.e. contain zero molecules of the starting material. Various hypotheses have been advanced to explain how a UD remedy might be different from unprepared solvent. One such hypothesis posits that a remedy contains stable clusters, i.e. localized regions where one or more hydrogen bonds remain fixed on a long time scale. High sensitivity proton nuclear magnetic resonance spectroscopy has not previously been used to look for evidence of differences between UD remedies and controls. METHODS: Homeopathic remedies made in water were studied via high sensitivity proton nuclear magnetic resonance spectroscopy. A total of 57 remedy samples representing six starting materials and spanning a variety of potencies from 6 C to 10 M were tested along with 46 controls. RESULTS: By presaturating on the water peak, signals could be reliably detected that represented H-containing species at concentrations as low as 5 microM. There were 35 positions where a discrete signal was seen in one or more of the 103 spectra, which should theoretically have been absent from the spectrum of pure water. Of these 35, fifteen were identified as machine-generated artifacts, eight were identified as trace levels of organic contaminants, and twelve were unexplained. Of the unexplained signals, six were seen in just one spectrum each. None of the artifacts or unexplained signals occurred more frequently in remedies than in controls, using a p < .05 cutoff. Some commercially prepared samples were found to contain traces of one or more of these small organic molecules: ethanol, acetate, formate, methanol, and acetone. CONCLUSION: No discrete signals suggesting a difference between remedies and controls were seen, via high sensitivity 1H-NMR spectroscopy. The results failed to support a hypothesis that remedies made in water contain long-lived non-dynamic alterations of the H-bonding pattern of the solvent.

Antivenom use in Australia. Premedication, adverse reactions and the use of venom detection kits.

OBJECTIVES: To analyse reports of antivenom use and sequelae in Australia from July 1 1989 to June 30 1990. The value of snake venom detection kits (VDKs) was also analysed. METHODS: Information was obtained from antivenom usage reports returned to the Commonwealth Serum Laboratories and from personal letters sent to those reporting doctors. Information on VDKs was obtained from antivenom usage reports or from questionnaires packaged with VDKs. RESULTS: Reported antivenoms used were: red-back spider, 258 cases; funnel-web spider, 3 cases; stonefish, 26 cases; box jellyfish, 6 cases; snake, 91 cases. Immediate reactions followed administration of red-back spider antivenom in only two patients and snake antivenoms in four patients. Delayed reactions (serum sickness) followed use of red-back spider antivenom in three patients, stonefish antivenom in two, and snake antivenoms in three. No reaction was life-threatening. Premedication was used in the majority of red-back spider bites and in 66 of 86 snake bites. Oral corticosteroids were given prophylactically after some uses of snake antivenom to prevent serum sickness. VDKs were used in 181 cases of snake bite and were reported as being useful in selecting appropriate snake antivenom in 31% of cases of antivenom use. (Only 10 of these 181 were also reported on the antivenom usage report.) Appropriate first aid was given in 61% of cases. There were 50% fewer snake bites reported than 10 years ago. CONCLUSIONS: Antivenoms in Australia are well tolerated with few immediate or delayed reactions. The use of premedication and prophylactic oral corticosteroids for four to five days after antivenom administration may be responsible for this low reaction rate. VDK results help select the appropriate antivenom; however, in some cases positive results were obtained from urine samples from patients with no symptoms of envenomation.

Helodermin-like peptides in thyroid C cells: stimulation of thyroid hormone secretion and suppression of calcium incorporation into bone.

Helodermin is a vasoactive intestinal peptide-like peptide in the salivary gland venom of the lizard Heloderma suspectum. Helodermin-like immunofluorescence was observed in the parafollicular (C) cells in several mammals and in the C cell homologues of the chicken ultimobranchial gland. Thus, helodermin-like peptides coexist with calcitonin. The results of radioimmunoassay agreed with the immunocytochemical findings. HPLC of rat thyroid extracts revealed one major peak of helodermin-like immunoreactivity, which eluted in a position close to that of lizard helodermin. Helodermin stimulated basal thyroid hormone secretion and colloid droplet formation in conscious mice. The effect of large doses of helodermin was quite long-lasting and the maximal response occurred after 2-6 hr. In addition, helodermin suppressed the incorporation of calcium into bone in conscious rats. The findings suggest that helodermin-like peptides in C cells may be involved in the local regulation of thyroid hormone secretion and in the maintenance of calcium homeostasis.

The Bela Schick Memorial Lecture. Toward measurement of success: from diphtheria to allergy.

Schick's skin test identifies persons immune to diphtheria and those successfully immunized. No test enables comparable judgment of hyposensitization (immunotherapy), long used for treatment of diseases of allergic origin. Efforts made to measure the successful immunologic conquest of diphtheria are compared and contrasted with efforts being made to conquer diseases of allergic origin.

Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions.

The aqueous extract of the venom glands of black widow spiders was fractionated on a column of Sephadex G-200 and then on a column of DEAE-Sephadex A-50 pH 8.2. A protein fraction was obtained that caused a great increase in the frequency of occurrence of miniature end plate potentials at the frog neuromuscular junction, and caused swelling of the nerve terminals and depleted them of their vesicles. The fraction consists of a least four protein components that are similar in their molecular weights (about 130,000) and isoelectric points (ranging from pH 5.2 to 5.5) and are immunologically indistinguishable. It contains no sugar residues and has little or no lipolytic or proteolytic activity. The fraction is toxic to mice and is different from the fractions that act on houseflies, the crayfish stretch receptor and the cockroach heart. It seems pure enough to warrant a detailed study of its site and mode of action.