Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.

Delayed-rectifier K؉ currents (I DR ) in pancreatic ␤-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltagegated K ؉ channel, K V 2.1, is expressed in ␤-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I DR in rodent ␤-cells. A novel peptidyl inhibitor of K V 2.1/K V 2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration ϳ1 nmol/l), has been purified, characterized, and used to probe the contribution of these channels to ␤-cell physiology. In mouse ␤-cells, GxTX-1 inhibits 90% of I DR and, as for K V 2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the ␤-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the ␤-cell I DR , which may provide advantages over currently used therapies for the treatment of type 2 diabetes.

Selective degradation reactions combined with MALDI analysis have been applied for molecular weight (MW) determination of polyether and polyester polyurethane (PUR) soft blocks. Selective degradation allows recovery of the polyols, and direct observation of the soft block oligomer distribution is possible for the first time by using MALDI. Ethanolamine is applied for polyether PUR degradation. MALDI analysis indicates that the recovered polytetrahydrofuran (pTHF) MW distribution is nearly identical to the unreacted pTHF material. Reduction in the ethanolamine reaction time allows observation of oligomer ions containing the diisocyanate linkage, which provide identification of the diisocyanate. Ethanolamine is not used for polyester PUR's degradation because the ester bonds will be cleaved. Therefore, phenylisocyanate is applied for polyester PUR degradation. Polybutylene adipate (pBA) oligomers were directly observed in the MALDI spectra of the degraded pBA-PUR samples. Comparison of the degraded pBA-PUR oligomer distribution with the unreacted pBA material indicates that low-mass oligomers are less abundant in the degraded pBA-PURs. Oligomer ions containing the diisocyanate linkage are also observed in the spectrum, providing a means for identifying the diisocyanate used for PUR syntheses. Size-exclusion chromatography (SEC) was combined with MALDI to provide accurate MW determination. Narrow MW fractions of the degraded and unreacted polyols were collected and analyzed by MALDI. This method allows precise calibration of the SEC chromatogram. The SEC-MALDI results provide significantly larger Mw and PD values than MALDI alone. Using SEC-MALDI, it was determined that the PD indexes of the pTHF and pBA samples are larger than the assumed values, which are based on the polyol synthesis reactions. The combination of selective degradation with SEC-MALDI, using either ethanolamine or phenylisocyanate, is a viable method for polyurethane polyol characterization.

A new TLC-MALDI direct coupling method which recovers approximately 100% of the analyte is presented. The method makes use of a hybrid TLC-MALDI plate in which a silica layer and a MALDI layer are configured adjacently on a common backing. After TLC separation, the plate is rotated 90 degrees and the separated analyte spots are eluted from the silica layer to the MALDI layer via capillary action of the MALDI layer. Signal-to-noise ratios are significantly improved over previously reported coupling methods. Low-femtomole detection limits have been demonstrated for small cyclic peptides, which are comparable to detection limits for standard MALDI measurements.

Quantification of a PEGylated peptide in human plasma using LC-MS/MS to support clinical studies presented challenges in terms of assay sensitivity, selectivity, and ruggedness. To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by two-dimensional (2D) LC-MS/MS was developed for MK-2662, an investigational peptide containing 38 amino acids with a 40 kDa branched PEG [poly(ethylene glycol)] at C-terminus. Biotinylated anti-PEG antibody, bound to streptavidin-coated magnetic beads, was used to capture MK-2662 and its stable-isotope-labeled internal standard from human plasma. After on-bead digestion with trypsin, the supernatant was injected on a 2D high-performance liquid chromatography (HPLC) system constructed with strong cation-exchange and reversed-phase columns, followed by MS/MS detection of the surrogate N(1-12)-mer of MK-2662 on an API5000. The assay ruggedness was improved by optimizing the trypsin digestion and sample storage conditions. The intraday validation, conducted in parallel with protein precipitation (PPT) assay, demonstrated 94.8-105.8% accuracy with <9.76% coefficient of variation (CV) for IAP, and 99.0-101.0% accuracy with <3.43% CV for PPT, over a dynamic range of 2-200 nM and 1-1000 nM, respectively. A cross comparison, performed using clinical samples, showed that the values obtained from IAP assay were about 15-30% lower than those from PPT method, which supports more specific PEG recognition provided by IAP.