Interfacing low-energy SAW nebulization with liquid chromatography-mass spectrometry for the analysis of biological samples

Soft ionization methods for the introduction of labile biomolecules into a mass spectrometer are of fundamental importance to biomolecular analysis. Previously, electrospray ionization (ESI) and matrix assisted laser desorption-ionization (MALDI) have been the main ionization methods used. Surface acoustic wave nebulization (SAWN) is a new technique that has been demonstrated to deposit less energy into ions upon ion formation and transfer for detection than other methods for sample introduction into a mass spectrometer (MS). Here we report the optimization and use of SAWN as a nebulization technique for the introduction of samples from a low flow of liquid, and the interfacing of SAWN with liquid chromatographic separation (LC) for the analysis of a protein digest. This demonstrates that SAWN can be a viable, low-energy alternative to ESI for the LC-MS analysis of proteomic samples

Mapping the interaction between eukaryotic initiation factor 4A (eIF4A) and the inhibitor hippuristanol using carbene footprinting and mass spectrometry

Protein-ligand interactions are central to protein activity and cell functionality. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in rational drug design towards the treatment of clinically relevant diseases. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that provides valuable information relating to protein-ligand interactions, such as the mapping of binding sites and associated conformational change. Here, we show the application of carbene footprinting to the interaction between eIF4A helicase and a natural product inhibitor, hippuristanol, found in the coral Isis hippuris. Upon addition of hippuristanol we identified reduced carbene labelling (masking) in regions of eIF4A previously implicated in ligand binding. Additionally, we detected hippuristanol-associated increased carbene labelling (unmasking) around the flexible hinge region of eIF4A, indicating ligand-induced conformational change. This work represents further development of the carbene footprinting technique and demonstrates its potential in characterising medicinally relevant protein-ligand interactions

Distinguishing Loss of Structure from Subunit Dissociation for Protein Complexes with Variable Temperature Ion Mobility Mass Spectrometry

The thermal stability and strength of interactions in proteins are commonly measured using isothermal calorimetry and differential scanning calorimetry providing a measurement that averages over structural transitions that occur as the proteins melt and dissociate. Here, we apply variable temperature ion mobility mass spectrometry (VT-IM-MS) to study the effect of temperature on the stability and structure of four multimeric protein complexes. VT-IM-MS is used here to investigate the change in the conformation of model proteins, namely, transthyretin (TTR), avidin, concanavalin A (conA), and human serum amyloid P component (SAP) at elevated temperatures prior, during, and after dissociation up to 550 K. As the temperature of the buffer gas is increased from 300 to 350 K, a small decrease in the collision cross sections (^DTCCS_He) of protein complexes from the values at room temperature is observed, and is associated with complex compaction occurring close to the reported solution <i>T</i>_m. At significantly higher temperatures, each protein complex undergoes an increase in ^DTCCS_He and in the width of arrival time distributions (ATD), which is attributed to extensive protein unfolding, prior to ejection of a highly charged monomer species. This approach allows us to decouple the distinct gas phase melting temperature (<i>T</i>_m) from the temperature at which we see subunit dissociation. The thermally induced dissociation (TID) mechanism is observed to initially proceed via the so-called “typical” (CID) dissociation route. Interestingly, data collected at higher analysis temperature suggests that the TID process might be adapting more “atypical” dissociation route

Profile and pro-health properties of Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni, z rodziny Asteraceae, pochodzi z Brazylii i Paragwaju. Wzbudziła zainteresowanie żywieniowców dzięki dwóm związkom: rebaudiozydowi A i stewiozydowi. Wymienione glikozydy mają właściwości słodzące do 300 razy silniejsze od sacharozy. Roślina charakteryzuje się również wysoką aktywnością przeciwutleniającą wynikającą m.in. z dużej zawartości polifenoli. Ekstrakty liści stewii wykazują korzystne działanie prozdrowotne. Wiąże się to ze specyficznym działaniem rośliny, obejmującym właściwości: przeciwzapalne, hipoglikemiczne, hipotensyjne, modulujące procesy immunologiczne, przeciwpróchnicze i przeciwwirusowe.Stevia rebaudiana Bertoni of the Asteraceae family originates from Brazil and Paraguay. It sparked interest among the nutritionists owing to its two compounds: rebaudioside A and stevioside. The latter glycosides have strong sweetening properties that are up to 300 times stronger than those of sucrose. The plant is also characterized by a high antioxidant activity resulting, among other things, from a high content of poly-phenols. Extracts of the stevia leaves demonstrate beneficial health effects. It is connected with the specific activity of this plant that comprises its following properties: anti-inflammatory, antihyperglycaemic, anti-hypertensive, immune system processes modelling ability, anti-cariogenic, and antiviral

Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements:Results from an Interlaboratory Comparison

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.</p