Detection of multiply charged protein ions using matrix-assisted laser desorption/ionization mass spectrometry and a force-dried droplet method with a 2-nitrophloroglucinol matrix

Conventional dried droplet (DD) methods show poor reproducibility in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) due to the frequent induction of a heterogeneous sample distribution. Recently, a forced dried droplet...

Multiplexed Conformationally-Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation

In this article, we present an approach for conformationally multiplexed localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-ion mobility-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. This arrangement was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome c. Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially-folded A-state. IM of deuterated ubiquitin 6+ ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5+ charge state showed greater protection against HDX than the compact 6+ conformer, and the 11+ charge state, ionized from conditions that stabilize the A-state, showed much greater deuterium incorporation. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with exterior and more unstructured regions of the protein, while interior, structured regions, especially those involved in heme binding, were more protected against exchange. These results, as well as potential future methods and experiments, are discussed herein.

EVALUATION OF HEPATOPROTECTIVE POTENTIALS OF METHANOL EXTRACT OF TEPHROSIA VILLOSA AGAINST THIOACETAMIDE INDUCED LIVER TOXICITY IN ALBINO RATS

Objective: The present study was conducted to determine the hepatoprotective potentials of methanol s extracts of Tephrosia villosa leaves against thioacetamide (TAA) induced liver damage in rats. Methodology: The acute oral toxicity study was conducted as per OECD guidelines, and the extract was proved to be safe up to the dose of 2000 mg/kg. The total duration of the study was 21 days, and animals were divided into six groups. Hepatotoxicity was induced in the animals of all groups except normal control by single dose administration of TAA (100 mg/kg) at 1st day of the study followed by animals were treated daily with standard drug silymarin and methanol extract of T. villosa (100 mg/kg, 200 mg/kg and 400 mg/kg) to respective groups for 21 days. Variations in biochemical parameters such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, direct bilirubin, albumin, total protein, ions and others parameters such as clotting time and weight of the liver were considered to determine beneficial effect of the extract. At the end of the study liver samples were collected and subjected to histopathological evaluation. Results: In control animals treated with TAA alone, there were variations in the above mentioned parameters. However in the animals treated with methanol extract and standard drug silymarin, all the parameters were normal possibly due to their beneficial property in protecting the liver against TAA induced hepatotoxicity. Conclusion: The results obtained in the above study suggesting that, the methanol extract of T. villosa possess significant hepatoprotective activity.

Evaluation of Hepatoprotective and in vivo Antioxidant Potentials of Methanol Extract of Tephrosia pumila against Thioacetamide Induced Liver Toxicity in Wistar Rats

The objective of the current investigation was performed to assess the hepatoprotective potentials and in vivo antioxidant properties of methanol extract of Tephrosia pumila against thioacetamide induced liver damage in rats. The acute oral toxicity study of methanol extract was determined as per OECD guidelines and the extract was proved to be safe up to the dose of 2000mg/kg. The total duration of the study was 21 days and animals were divided into six groups. Hepatotoxicity was induced in the animals of all groups except normal control by single dose administration of Thioacetamide(100mg/kg) at first day of the study followed by animals were treated daily with standard drug sylimarin and methanol extract of Tephrosia pumila (100mg/kg, 200mg/kg and 400mg/kg) to respective groups for 21 days. Variations in biochemical parameters like alanine transferase (ALT), aspartate transferase (AST), alkaline phosphatase (ALP), total bilirubin, direct bilirubin, albumin, total protein, ions and others parameters like clotting time and weight of the liver were considered to determine beneficial effect of the extract. At the end of the study liver samples were collected and subjected to histopathological evaluation. There were significant variations in the above mentioned biochemical parameters in toxic control animals treated with Thioacetamide alone while in the animals treated with methanol extract and standard drug silymarin, all the parameters were normal possibly due to their beneficial property in protecting the liver against thioacetamide induced hepatotoxicity. The results obtained in the above study suggesting that, the methanol extract of Tephrosia pumila possess significant hepatoprotective activity.

Proteoform-selective imaging of tissues using mass spectrometry

Unraveling the complexity of biological systems relies on the development of new approaches for spatially resolved proteoform-specific analysis of the proteome. Top-down proteomics is a powerful tool, which has been used for the identification of thousands of proteoforms in biological samples. Herein, we present a first spatially resolved top-down proteomics analysis of biological tissues using nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI). Nano-DESI generates multiply charged protein ions, which is advantageous for their structural characterization using tandem mass spectrometry (MS/MS). Proof-of-concept experiments demonstrate that the nano-DESI MSI combined with on-tissue top-down proteomics is ideally suited for the proteoform-selective imaging of thin tissue sections. Using rat brain tissue as a model system, we provide the first evidence of the differential proteoform expression in different regions of the brain.