Peptides injection sensitivity

Another potentially paralytic conotoxin was recently described this was a peptide purified from Conus geographus venom, which like / -conotoxin appeared to target to voltage-sensitive Na channels. However, the structure of "conotoxin GS" [nomenclature of Yanagawa et al. (J7)] was less homologous to / -conotoxins than to the w-conotoxins, which are Ca channel blockers. The same peptide was purified and characterized using a different assay, the induction of highly aberrant behavior upon ic injection of mice (L. J. Cruz, unpublished data). 

Erythromycin, a macrolide antibiotic, lacks a significant chromophore. Detection sensitivity was enhanced by using a wavelength of 200 nm and selecting an injection solvent of lower conductivity than the BGE. In order to facilitate the separation of erythromycin and its related substances, 35% (v/v) ethanol was incorporated into a 150 mM phosphate buffer pH 7.5. Resolution of all of the compounds was achieved in approximately 45 min. The method was employed as an assay method for erythromycin and for impurity determination. Peptide antibiotics, such as colistin and polymyxin, are mixtures of many closely related compounds. A validated CZE method for impurity analysis of polymyxin B was described, employing 130 mM triethanolamine-phosphate buffer at pH 2.5 to reduce the adsorption of analyte onto the capillary wall. Methyl-/l-cyclodextrin (M-/1-CD) and 2-propanol were found to be necessary for selectivity enhancement. Using similar buffer additives, the same group developed and validated a method for colistin analysis. ... 

This test is also very sensitive to treatments that increase anxiety, such as injections of peptides like corticotropinreleasing factor, which is known to cause the release of stress hormones and produce anxiety. 

When CGRP is injected into the central nervous system, it produces a variety of effects, including hypertension and suppression of feeding. When injected into the systemic circulation, the peptide causes hypotension and tachycardia. The hypotensive action of CGRP results from the potent vasodilator action of the peptide indeed, CGRP is the most potent vasodilator yet discovered. It dilates multiple vascular beds, but the coronary circulation is particularly sensitive. 

Once it was established that pheromone biosynthesis was regulated by a peptide produced in the SEG, the next goal was to identify the peptide. In the purification of any biologically active factor, each purification step requires a sensitive bioassay to measure the active material. In the purification of PBAN, the bioassay consisted of head ligated females that were injected with bioactive fractions. After a 1-3 h period of incubation, the pheromone gland was excised and titers of pheromone determined by gas chromatography (GC). The first PBAN was purified and identified from H. zea (Raina el ah, 1989). Dissection of about 5000 brain-SEG complexes followed by several steps of HPLC purification resulted in a pure peptide that could be sequenced. It was found to be a 33 amino acid peptide with a C-terminal amide (Table 5.1). The peptide was synthesized and was shown to be active in the bioassay in a dose as low as 2 pmol (Raina et al., 1989). In the same year, a PBAN from B. mori was purified and sequenced (Kitamura et al.,... 

Due to the high sensitivity it is favorably to couple a nanoHPLC to an ESI-source. As mass spectrometers are concentration dependent detectors, the sensitivity of an instrumental setup is mostly determined by the peptide concentration of the eluate but not by the peptide amount. Thus a nanocolumn with a flow rate of 300 nL/min provides an about thousand times higher sensitivity than a microbore column with a flow rate of 300 (xL/min. As an alternative to buying a nanoHPLC system it is also possible to use a relatively inexpensive flow splitter after the pump and before the injection valve and the column. Thereby the flow rate can be reduced to use a capillary column (flow rate 4 (xL/min) on an analytical HPLC system or a nanocolumn on a capillary HPLC system. Instead of a flow-splitter it is preferred to couple a nanoHPLC to an ESI-source. Thereby, the flow rate is split according to the column backpressure, i.e., mostly the column volumes if the same packing materials are used. However, these low-cost setups are less reliable than a nanoHPLC and the reproducibility is worse. 

Figure 6.4 Comparison of sensitivities for different sample injection techniques using a 50 mg/ml peptide mixture (a) hydrostatic injection, (b) electromigration injection where the sample is dissolved in buffer, and (c) electromigration injection where the sample is dissolved in water. (Adapted from Ref. 1 with permission.)...

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