Biology subjects

Ivanov, A. R.

Publications and source records attributed to Ivanov, A. R..

3 recordsLinked to original sources

bioRxiv2023

Implementation of an enriched membrane protein carrier channel for enhanced detection of membrane proteins in mass spectrometry-based thermal stability assays

In this work, we developed a membrane-enriched stable isotope isobaric-labeled carrier channel (meSIILCC) for mass spectrometry-based thermal stability assay (MS-TSA). A proof-of-concept study demonstrated that the meSIILCC method could modestly improve membrane protein (MP) detection in MS-TSA experiments. An enhancement of 10% in identifications of membrane-proteins was observed in the meSIILCC group. Hydrophobicity analysis of the identified and quantified peptides using the grand average of hydropathy index confirmed the meSIILCC approach enriched for peptides of higher hydrophobicity characteristic of membrane-associated proteins. To further improve meSIILCC, four membrane-protein enrichment approaches were compared. Using the selected and optimized workflow that utilized isobaric labeling-mass spectrometry, 8,662 protein groups were quantitatively characterized and then annotated based on their subcellular localization. The corresponding reporter ion intensities were used to construct a heatmap, which revealed an increased representation of proteins corresponding to the "plasma membrane" gene ontology term. In a separate DMSO-only MS-TSA experiment, the optimally performing meSIILCC was added at 10-fold the protein content of the lowest heated aliquot from the MS-TSA, and isotope interference was found to be the highest in the 134N channel, while to a much lesser degree in other channels that were left empty. To further assess the performance of meSIILCC in the DMSO-only MS-TSA experiment, an over-representation analysis was performed, which demonstrated that proteins exclusive to the meSIILCC group had more than a five-fold increase in gene ontology cellular component terms related to the "membrane" term. We found 496 proteins from the DMSO-only MS-TSA experiment, which were identified across all replicates and shared between the meSIILCC and control that were annotated with "plasma membrane." A close to 28% increase in the set corresponding to unique peptides was realized, using the meSIILCC approach, with a median value of 6.3 peptides per protein, compared to 4.7 in the control.

biochemistry↗

bioRxiv2020

A simple, high-throughput method of protein and label removal from extracellular vesicle samples

Evidence continues to increase of the clinical utility extracellular vesicles (EVs) can provide as translational biomarkers. While a wide variety of EV isolation and purification methods have been implemented, few techniques are high-throughput and scalable for removing excess fluorescent reagents (e.g. dyes, antibodies). EVs are too small to be recovered from routine cell-processing procedures, such as filtration or centrifugation. The lack of suitable methods for removing unbound labels, especially in optical assays, is a major roadblock to accurate EV phenotyping and utilization of EV assays in a translational or clinical setting. Therefore, we developed a method for using a multi-modal resin, referred to as EV-Clean, to remove unbound labels from EV samples, and we demonstrate improvement in flow cytometric EV analysis with the use of this EV-Clean method.

biochemistry↗

bioRxiv2019

Channeling macrophage polarization by using a selective translation inhibitor

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatement of primary macrophages with rocaglates increased their resilience to oxidative stress, stimulated autophagy and killing of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization towards the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g. chronic bacterial infections, allergies and, possibly, certain tumors.

immunology↗