Biology subjects

Ashkarran, A. A.

Publications and source records attributed to Ashkarran, A. A..

7 recordsLinked to original sources

bioRxiv2026

High-Sensitivity Magnetic Levitation Reveals Intrinsic Protein Corona Heterogeneity on Identical Nanoparticles

The protein corona (PC), a layer of biomolecules that adsorbs onto nanoparticles (NPs) surfaces upon exposure to biological fluids, plays a key role in defining the biological identity and performance of nanomaterials. However, most current analytical approaches rely on pooled measurements of PC-coated NPs and therefore lack the resolution needed to detect subtle heterogeneity in PC composition, potentially masking important differences in NPs biological identity. Here, we used a high-sensitivity magnetic levitation (MagLev) platform capable of resolving extremely small density differences among nominally identical PC-coated NPs, enabling fractionation of particles based on subtle variations in PC composition. Compared with conventional standard MagLev systems (density resolution [~]10-3 g/cm3), the high-sensitivity MagLev improves density sensitivity by up to three orders of magnitude, allowing discrimination of density differences as small as 10-5 g/cm3. Using this approach, PC-coated NPs were separated along the MagLev column into multiple fractions corresponding to distinct density populations. Subsequent proteomic analysis across the extracted fractions identified more than 500 proteins and revealed a structured but continuous redistribution of protein composition across the column, including fraction-dependent differences in protein abundance, overlap, and biological identity. In particular, the fraction series captured hidden heterogeneity among nominally identical PC-coated NPs, with upper fractions retaining relatively stronger extracellular/plasma-associated signatures and lower fractions showing increasing representation of structural, membrane-associated, cytoskeletal, and metabolic proteins. These findings demonstrate that PC formation is intrinsically heterogeneous even on identical NPs and this heterogeneity is largely missed by conventional pooled analysis. High-sensitivity MagLev provides a simple, label-free framework for resolving PC heterogeneity and offers a new analytical approach for studying NP-biomolecule interactions, with important implications for nanomedicine design, biomarker discovery, and the clinical translation of NP-based systems. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/727410v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@155ad62org.highwire.dtl.DTLVardef@1ea3e3dorg.highwire.dtl.DTLVardef@19c40ceorg.highwire.dtl.DTLVardef@162ca8d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Magnetic Levitation Derived Metabolomic Fingerprinting Enables Exploratory Discrimination of Breast Cancer Subtypes

Breast cancer (BC) consists of heterogeneous molecular subtypes with distinct biological behavior and therapeutic response, including triple negative breast cancer (TNBC), human epidermal growth factor receptor 2 positive (HER2+), and Luminal A tumors. Current subtype classification primarily relies on tissue biopsy and molecular pathology, highlighting the need for complementary non invasive analytical approaches capable of capturing systemic biochemical differences among BC subtypes. In this exploratory study, we investigate whether magnetic levitation (MagLev) derived plasma patterning combined with untargeted metabolomics analysis can distinguish BC subtypes based on subtype specific metabolomic fingerprints. Representative plasma samples from TNBC, HER2+, and Luminal A patients were levitated in a standard MagLev system containing superparamagnetic iron oxide nanoparticles (SPIONs), generating visibly distinct levitation patterns throughout the levitation process. Individual levitated plasma layers were subsequently extracted and subjected to untargeted metabolomics analysis. Multivariate analysis demonstrated clear subtype dependent metabolic separation among the three BC subtypes. Heatmap clustering, PLS DA analysis, and variable importance profiling identified distinct metabolic signatures involving glycolysis, pentose phosphate pathway metabolism, TCA cycle activity, amino acid metabolism, and lipid remodeling. Elevated levels of phosphoglycerate, pyruvate, ribose 5 phosphate, glutamate, and 2 oxoglutarate suggested enhanced proliferative and biosynthetic metabolism, while enrichment of long chain fatty acids in Luminal A samples indicated subtype specific lipid metabolic remodeling. These findings demonstrate the feasibility of combining MagLev derived plasma organization with metabolomics analysis to generate disease specific metabolomic fingerprints and establish a foundation for future large scale validation studies.

biophysics↗

bioRxiv2026

MXene Protein Corona Interfaces for Molecular Profiling of Alzheimers Disease

The protein corona (PC) that forms on the surface of nanomaterials upon contact with biological fluids provides a molecular snapshot of the hosts physiological and pathological state. Here, we investigate two-dimensional (2D) titanium carbide (Ti3C2Tx) MXene nanosheets as nanobiointerfaces for capturing Alzheimers disease (AD)-associated plasma protein signatures. Ti3C2Tx MXene flakes were incubated with plasma from clinically diagnosed AD patients and age-matched healthy controls (HC), leading to the formation of Ti3C2Tx MXene-PC complexes. Physicochemical characterization using dynamic light scattering, zeta potential analysis, and transmission electron microscopy revealed disease-dependent changes in hydrodynamic size, surface charge, and PC profile. Proteomic analysis of the isolated PC layers quantified 1,611 proteins without prior fractionation, demonstrating effective enrichment of low-abundance plasma components. Principal component analysis (PCA) revealed consistent separation between AD- and HC-derived Ti3C2Tx MXene-PC proteomes despite inter-individual heterogeneity. Differential abundance analysis identified selective enrichment of heterogeneous nuclear ribonucleoproteins (hnRNPs), annexins, and inflammatory mediators in AD-derived PC, implicating dysregulated RNA metabolism, membrane stress responses, and immune activation, hallmark processes in AD pathology. Our findings demonstrate that Ti3C2Tx MXene-PC interfaces act as selective molecular filters that reshape the detectable plasma proteome, enabling disease-associated molecular phenotyping and establishing a versatile nanointerface-driven framework for uncovering AD-related plasma signatures, providing a foundation for future translational diagnostic development.

biophysics↗

bioRxiv2024

Standardizing protein corona characterization in nanomedicine: a multi-center study to enhance reproducibility and data homogeneity

Our recent findings reveal substantial variability in the characterization of identical protein corona across different proteomics facilities, demonstrating that protein corona datasets are not easily comparable between independent studies. We have shown that heterogeneity in the final composition of the identical protein corona mainly originates from variations in sample preparation protocols, liquid chromatography mass spectrometry (LC-MS) workflows, and raw data processing. Here, to address this issue, we developed standardized protocols and unified sample preparation workflows, and distributed identical protein corona digests to several proteomics centers that performed better in our previous study. Additionally, we examined the influence of using similar mass spectrometry instruments on data homogeneity. Furthermore, we evaluated whether standardizing database search parameters and data processing workflows could enhance data uniformity. More specifically, our new findings reveal a remarkable, stepwise improvement in protein corona data consistency across various proteomics facilities. Streamlining the whole workflow results in a dramatic increase in protein ID overlaps from 11% for good centers to 40% across core facilities that utilized similar instruments and were subjected to a uniform database search. This comprehensive analysis identifies key factors contributing to data heterogeneity in mass spectrometry-based proteomics of protein corona and plasma-related samples. By streamlining these processes, our findings significantly advance the potential for consistent and reliable nanomedicine-based diagnostics and therapeutics across different studies.

bioengineering↗

bioRxiv2024

Mass spectrometry-based top-down proteomics in nanomedicine: proteoform-specific measurement of protein corona

Conventional mass spectrometry (MS)-based bottom-up proteomics (BUP) analysis of protein corona [i.e., an evolving layer of biomolecules, mostly proteins, formed on the surface of nanoparticles (NPs) during their interactions with biomolecular fluids] enabled nanomedicine community to partly identify the biological identity of NPs. Such an approach, however, fails pinpoint the specific proteoforms--distinct molecular variants of proteins, which is essential for prediction of the biological fate and pharmacokinetics of nanomedicines. Recognizing this limitation, this study pioneers a robust and reproducible MS-based top-down proteomics (TDP) technique for precisely characterizing proteoforms in the protein corona. Our TDP approach has successfully identified hundreds of proteoforms in the protein corona of polystyrene NPs, ranging from 3-70 kDa, revealing over 20 protein biomarkers with combinations of post-translational modifications, signal peptide cleavages, and/or truncations--details that BUP could not fully discern. This advancement in MS-based TDP offers a more comprehensive and exact characterization of NP protein coronas, deepening our understanding of NPs biological identities and potentially revolutionizing the field of nanomedicine.

systems biology↗

bioRxiv2024

Deep Plasma Proteome Profiling by Modulating Single Nanoparticle Protein Corona with Small Molecules

The protein corona, a dynamic biomolecular layer that forms on nanoparticle (NP) surfaces upon exposure to biological fluids is emerging as a valuable diagnostic tool for improving plasma proteome coverage analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Here, we show that spiking small molecules, including metabolites, lipids, vitamins, and nutrients (namely, glucose, triglyceride, diglycerol, phosphatidylcholine, phosphatidylethanolamine, L--phosphatidylinositol, inosine 5'-monophosphate, and B complex), into plasma can induce diverse protein corona patterns on otherwise identical NPs, significantly enhancing the depth of plasma proteome profiling. The protein coronas on polystyrene NPs when exposed to plasma treated with an array of small molecules (n=10) allowed for detection of 1793 proteins marking an 8.25-fold increase in the number of quantified proteins compared to plasma alone (218 proteins) and a 2.63-fold increase relative to the untreated protein corona (681 proteins). Furthermore, we discovered that adding 1000 {micro}g/ml phosphatidylcholine could singularly enable the detection of 897 proteins. At this specific concentration, phosphatidylcholine selectively depleted the four most abundant plasma proteins, including albumin, thus reducing the dynamic range of plasma proteome and enabling the detection of proteins with lower abundance. By employing an optimized data-independent acquisition (DIA) approach, the inclusion of phosphatidylcholine led to the detection of 1436 proteins in a single plasma sample. Our molecular dynamic results revealed that phosphatidylcholine interacts with albumin via hydrophobic interactions, h-bonds, and water-bridges. Addition of phosphatidylcholine also enabled the detection of 337 additional proteoforms compared to untreated protein corona using a top-down proteomics approach. These significant achievements are made utilizing only a single NP type and one small molecule to analyze a single plasma sample, setting a new standard in plasma proteome profiling. Given the critical role of plasma proteomics in biomarker discovery and disease monitoring, we anticipate widespread adoption of this methodology for identification and clinical translation of proteomic biomarkers into FDA approved diagnostics.

bioengineering↗

bioRxiv2022

Multi-Omics Analysis of Magnetically Levitated Plasma Biomolecules

We recently discovered that superparamagnetic iron oxide nanoparticles (SPIONs) can levitate plasma biomolecules in the magnetic levitation (MagLev) system and cause formation of ellipsoidal biomolecular bands. To better understand the composition of the levitated biomolecules in various bands, we comprehensively characterized them by multi-omics analyses. To probe whether the biomolecular composition of the levitated ellipsoidal bands correlates with the health of plasma donors, we used plasma from individuals who had various types of multiple sclerosis (MS), as a model disease with significant clinical importance. Our findings reveal that, while the composition of proteins does not show much variability, there are significant differences in the lipidome and metabolome profiles of each magnetically levitated ellipsoidal band. By comparing the lipidome and metabolome compositions of various plasma samples, we found that the levitated biomolecular ellipsoidal bands do contain information on the health status of the plasma donors. More specifically, we demonstrate that there are particular lipids and metabolites in various layers of each specific plasma pattern that significantly contribute to the discrimination of different MS subtypes, i.e., relapsing-remitting MS (RRMS), secondary-progressive MS (SPMS), and primary-progressive MS (PPMS). These findings will pave the way for utilization of MagLev of biomolecules in biomarker discovery and diagnosis of this and other complex disorders.

bioengineering↗