Standardizing Protein Corona Characterization in Nanomedicine: A Multicenter Study to Enhance Reproducibility and Data Homogeneity.

We recently revealed significant variability in protein corona characterization across various proteomics facilities, indicating that data sets are not comparable between independent studies. This heterogeneity mainly arises from differences in sample preparation protocols, mass spectrometry workflows, and raw data processing. To address this issue, we developed standardized protocols and unified sample preparation workflows, distributing uniform protein corona digests to several top-performing proteomics centers from our previous study. We also examined the influence of using similar mass spectrometry instruments on data homogeneity and standardized database search parameters and data processing workflows. Our findings reveal a remarkable stepwise improvement in protein corona data uniformity, increasing overlaps in protein identification from 11% to 40% across facilities using similar instruments and through a uniform database search. We identify the key parameters behind data heterogeneity and provide recommendations for designing experiments. Our findings should significantly advance the robustness of protein corona analysis for diagnostic and therapeutics applications.

The role of protein corona in advancing plasma proteomics.

The protein corona, a layer of biomolecules forming around nanoparticles in biological environments, critically influences nanoparticle interactions with biosystems, affecting pharmacokinetics and biological outcomes. Initially, the protein corona presented challenges for nanomedicine and nanotoxicology, such as nutrient depletion in cell cultures and masking of nanoparticle-targeting species. However, recent advancements have highlighted its potential in environmental toxicity, proteomics, and immunology. This viewpoint focuses on leveraging the protein corona to enhance the depth of plasma proteome analysis, addressing challenges posed by the high dynamic range of protein concentrations in plasma. The protein corona simplifies sample preparation, enriches low-abundance proteins, and improves proteome coverage. Innovations include using diverse nanoparticles and spiking small molecules to increase the number of quantified proteins. Reproducibility issues across core facilities necessitate standardized protocols. Moreover, top-down proteomics enables proteoform-specific measurements, providing deeper insights into protein corona composition. Future research should aim at improving top-down proteomics techniques and integrating protein corona studies and proteomics for personalized medicine and advanced diagnostics.

Gel-Assisted Proteome Position Integral Shift Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Caspase 3 Substrates.

Here, we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics and demonstrates its utility in studying proteolytic events in programmed cell death. With gel-assisted proteome position integral shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: (i) a negative MW shift; (ii) an elevated ratio in a pair of a semitryptic and tryptic peptide, (iii) a negative shift in the standard deviation of MW estimated for different peptides, and (iv) a negative shift in skewness of the same data. Of these proteins, 58 molecules were previously unreported caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, the GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.

Massive Solubility Changes in Neuronal Proteins upon Simulated Traumatic Brain Injury Reveal the Role of Shockwaves in Irreversible Damage.

We investigated the immediate molecular consequences of traumatic brain injuries (TBIs) using a novel proteomics approach. We simulated TBIs using an innovative laboratory apparatus that employed a 5.1 kg dummy head that held neuronal cells and generated a ≤4000 g-force acceleration upon impact. A Proteome Integral Solubility Alteration (PISA) assay was then employed to monitor protein solubility changes in a system-wide manner. Dynamic impacts led to both a reduction in neuron viability and massive solubility changes in the proteome. The affected proteins mapped not only to the expected pathways, such as those of cell adhesion, collagen, and laminin structures, as well as the response to stress, but also to other dense protein networks, such as immune response, complement, and coagulation cascades. The cellular effects were found to be mainly due to the shockwave rather than the g-force acceleration. Soft materials could reduce the impact's severity only until they were fully compressed. This study shows a way of developing a proteome-based meter for measuring irreversible shockwave-induced cell damage and provides a resource for identifying protein biomarkers of TBIs and potential drug targets for the development of products aimed at primary prevention and intervention.

Abnormal (Hydroxy)proline Deuterium Content Redefines Hydrogen Chemical Mass.

Analyzing the δ2H values in individual amino acids of proteins extracted from vertebrates, we unexpectedly found in some samples, notably bone collagen from seals, more than twice as much deuterium in proline and hydroxyproline residues than in seawater. This corresponds to at least 4 times higher δ2H than in any previously reported biogenic sample. We ruled out diet as a plausible mechanism for such anomalous enrichment. This finding puts into question the old adage that "you are what you eat".

Proteomics-Compatible Fourier Transform Isotopic Ratio Mass Spectrometry of Polypeptides.

Measuring the relative abundances of heavy stable isotopes of the elements C, H, N, and O in proteins is of interest in environmental science, archeology, zoology, medicine, and other fields. The isotopic abundance measurements of the fine structure of immonium ions with ultrahigh resolution mass spectrometry obtained in gas-phase fragmentation of polypeptides have previously uncovered anomalous deuterium enrichment in (hydroxy)proline of bone collagen in marine mammals. Here, we provide a detailed description and validation of this approach and demonstrate per mil-range precision of isotopic ratio measurements in aliphatic residues from proteins and cell lysates. The analysis consists of proteomics-type experiment demanding sub-microgram amounts of a protein sample and providing concomitantly protein sequence data allowing one to verify sample purity and establish its identity. A novel software tool protein amino acid-resolved isotopic ratio mass spectrometry (PAIR-MS) is presented for extracting isotopic ratio data from the raw data files acquired on an Orbitrap mass spectrometer.

First Experimental Evidence for Reversibility of Ammonia Loss from Asparagine.

Ammonia loss from L-asparaginyls is a nonenzymatic reaction spontaneously occurring in all proteins and eventually resulting in damaging isoaspartate residues that hamper protein function and induce proteinopathy related to aging. Here, we discuss theoretical considerations supporting the possibility of a full repair reaction and present the first experimental evidence of its existence. If confirmed, the true repair of L-asparaginyl deamidation could open new avenues for preventing aging and neurodegenerative diseases.

COVID-19: Nanomedicine Uncovers Blood-Clot Mystery.

Further complications associated with infection by severe acute respiratory syndrome coronavirus 2 (a.k.a. SARS-CoV-2) continue to be reported. Very recent findings reveal that 20-30% of patients at high risk of mortality from COVID-19 infection experience blood clotting that leads to stroke and sudden death. Timely assessment of the severity of blood clotting will be of enormous help to clinicians in determining the right blood-thinning medications to prevent stroke or other life-threatening consequences. Therefore, rapid identification of blood-clotting-related proteins in the plasma of COVID-19 patients would save many lives. Several nanotechnology-based approaches are being developed to diagnose patients at high risk of death due to complications from COVID-19 infections, including blood clots. This Perspective outlines (i) the significant potential of nanomedicine in assessing the risk of blood clotting and its severity in SARS-CoV-2 infected patients and (ii) its synergistic roles with advanced mass-spectrometry-based proteomics approaches in identifying the important protein patterns that are involved in the occurrence and progression of this disease. The combination of such powerful tools might help us understand the clotting phenomenon and pave the way for development of new diagnostics and therapeutics in the fight against COVID-19.

Comparative Proteomics of Dying and Surviving Cancer Cells Improves the Identification of Drug Targets and Sheds Light on Cell Life/Death Decisions.

Chemotherapeutics cause the detachment and death of adherent cancer cells. When studying the proteome changes to determine the protein target and mechanism of action of anticancer drugs, the still-attached cells are normally used, whereas the detached cells are usually ignored. To test the hypothesis that proteomes of detached cells contain valuable information, we separately analyzed the proteomes of detached and attached HCT-116, A375, and RKO cells treated for 48 h with 5-fluorouracil, methotrexate and paclitaxel. Individually, the proteomic data on attached and detached cells had comparable performance in target and drug mechanism deconvolution, whereas the combined data significantly improved the target ranking for paclitaxel. Comparative analysis of attached versus detached proteomes provided further insight into cell life and death decision making. Six proteins consistently up- or downregulated in the detached versus attached cells regardless of the drug and cell type were discovered; their role in cell death/survival was tested by silencing them with siRNA. Knocking down USP11, CTTN, ACAA2, and EIF4H had anti-proliferative effects, affecting UHRF1 additionally sensitized the cells to the anticancer drugs, while knocking down RNF-40 increased cell survival against the treatments. Therefore, adding detached cells to the expression proteomics analysis of drug-treated cells can significantly increase the analytical value of the approach. The data have been deposited to the ProteomeXchange with identifier PXD007686.

Dynamic Proteomics Reveals High Plasticity of Cellular Proteome: Growth-Related and Drug-Induced Changes in Cancer Cells are Comparable.

In chemical proteomics, the changes occurring in cellular proteomes upon drug treatment are used to identify the drug targets and the mechanism of action. However, proteomes of cultured cells undergo also natural alteration associated with changes in the media, attaining a degree of confluence as well as due to cell division and cell metabolism. These changes are implicitly assumed to be smaller in magnitude than the drug-induced changes that ultimately lead to cell demise. In this study, it is shown that growth-related proteome changes in the untreated control group are comparable in magnitude to drug-induced changes over the course of 48 h treatment. In two well-characterized cancer cell lines, growth-related effects assessed with deep proteomics analysis (10 481 proteins quantified with at least two peptides) show common trends, the steady downregulation of cell division processes, and the upregulation of metabolism-related pathways. The magnitude of these variations, which are present even before reaching 100% confluence reveals unexpectedly high plasticity of the cellular proteome. This finding reinforces the need, generally accepted in theory but not always followed in practice, to use a time-matched control when measuring drug-induced proteome changes.

Microparticles containing erlotinib-loaded solid lipid nanoparticles for treatment of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) patients with sensitizing mutations in the exons 18-21 of the epithelial growth factor receptor (EGFR) gene show increased kinase activity of EGFR. Hence, tyrosine kinase inhibitors (TKIs) such as erlotinib (ETB) have commonly been used as the second line therapeutic option for the treatment of metastatic NSCLC. While the ETB is available as an oral dosage form, the local delivery of this TKI to the diseased cells of the lung may ameliorate its therapeutic impacts. In the current study, we report on the development of ETB-loaded solid lipid nanoparticle (SLN) based formulation of dry powder inhaler (ETB-SLN DPI). ETB-SLNs were formulated using designated amount of compritol/poloxamer 407. The engineered ETB-SLNs showed sub-100 nm spherical shape with an encapsulation efficiency of 78.21%. MTT assay and DAPI staining revealed that the ETB-SLNs enhanced the cytotoxicity of cargo drug molecules in the human alveolar adenocarcinoma epithelial A549 cells as a model for NSCLC. To attain the ETB-SLN DPI, the ETB-SLNs were efficiently spray dried into microparticles (1-5 µm) along with mannitol. The ETB-SLN DPI powder displayed suitable flowability and aerodynamic traits. The Carr's Index, Hausner ratio and Next Generation Impactor (NGI) analyses confirmed deep inhalation pattern of the formulation. Based on these findings, we propose the ETB-SLN DPI as a promising treatment modality for the NSCLC patients.

Targeted superparamagnetic iron oxide nanoparticles for early detection of cancer: Possibilities and challenges.

Nanomedicine, the integration of nanotechnological tools in medicine demonstrated promising potential to revolutionize the diagnosis and treatment of various human health conditions. Nanoparticles (NPs) have shown much promise in diagnostics of cancer, especially since they can accommodate targeting molecules on their surface, which search for specific tumor cell receptors upon injection into the blood stream. This concentrates the NPs in the desired tumor location. Furthermore, such receptor-specific targeting may be exploited for detection of potential metastases in an early stage. Some NPs, such as superparamagnetic iron oxide NPs (SPIONs), are also compatible with magnetic resonance imaging (MRI), which makes their clinical translation and application rather easy and accessible for tumor imaging purposes. Furthermore, multifunctional and/or theranostic NPs can be used for simultaneous imaging of cancer and drug delivery. In this review article, we will specifically focus on the application of SPIONs in early detection and imaging of major cancer types. FROM THE CLINICAL EDITOR: Super-paramagnetic iron oxide nanoparticles (SPIONs) have been reported by many to be useful as an MRI contrast agent in the detection of tumors. To further enhance the tumor imaging, SPIONs can be coupled with tumor targeting motifs. In this article, the authors performed a comprehensive review on the current status of using targeted SPIONS in tumor detection and also the potential hurdles to overcome.

Chemical Proteomics Reveals that the Anticancer Drug Everolimus Affects the Ubiquitin-Proteasome System.

Rapamycin is a natural antifungal, immunosuppressive, and antiproliferative compound that allosterically inhibits mTOR complex 1. The ubiquitin-proteasome system (UPS) responsible for protein turnover is usually not listed among the pathways affected by mTOR signaling. However, some previous studies have indicated the interplay between the UPS and mTOR. It has also been reported that rapamycin and its analogs can allosterically inhibit the proteasome itself. In this work, we studied the molecular effect of rapamycin and its analogs (rapalogs), everolimus and temsirolimus, on the A549 cell line by expression proteomics. The analysis of differentially expressed proteins showed that the cellular response to everolimus treatment is strikingly different from that to rapamycin and temsirolimus. In the cluster analysis, the effect of everolimus was similar to that of bortezomib, a well-established proteasome inhibitor. UPS-related pathways were enriched in the cluster of proteins specifically upregulated upon everolimus and bortezomib treatments, suggesting that both compounds have similar proteasome inhibition effects. In particular, the total amount of ubiquitin was significantly elevated in the samples treated with everolimus and bortezomib, and analysis of the polyubiquitination patterns revealed elevated intensities of the ubiquitin peptide with a GG modification at the K48 residue, consistent with a bottleneck in proteasomal protein degradation. Moreover, the everolimus treatment resulted in both ubiquitin phosphorylation and generation of a significant amount of semitryptic peptides, illustrating the increase in the protease activity. These observations suggest that everolimus affects the UPS in a unique way, and its mechanism of action is different from that of its close chemical analogs, rapamycin and temsirolimus.

A uniform data processing pipeline enables harmonized nanoparticle protein corona analysis across proteomics core facilities.

Protein corona, a layer of biomolecules primarily comprising proteins, forms dynamically on nanoparticles in biological fluids and is crucial for predicting nanomedicine safety and efficacy. The protein composition of the corona layer is typically analyzed using liquid chromatography-mass spectrometry (LC-MS/MS). Our recent study, involving identical samples analyzed by 17 proteomics facilities, highlighted significant data variability, with only 1.8% of proteins consistently identified across these centers. Here, we implement an aggregated database search unifying parameters such as variable modifications, enzyme specificity, number of allowed missed cleavages and a stringent 1% false discovery rate at the protein and peptide levels. Such uniform search dramatically harmonizes the proteomics data, increasing the reproducibility and the percentage of consistency-identified unique proteins across distinct cores. Specifically, out of the 717 quantified proteins, 253 (35.3%) are shared among the top 5 facilities (and 16.2% among top 11 facilities). Furthermore, we note that reduction and alkylation are important steps in protein corona sample processing and as expected, omitting these steps reduces the number of total quantified peptides by around 20%. These findings underscore the need for standardized procedures in protein corona analysis, which is vital for advancing clinical applications of nanoscale biotechnologies.

Adding Color to Mass Spectra of Biopolymers: Charge Determination Analysis (CHARDA) Assigns Charge State to Every Ion Peak.

Traditionally, mass spectrometry (MS) output is the ion abundance plotted versus the ionic mass-to-charge ratio m/z. While employing only commercially available equipment, Charge Determination Analysis (CHARDA) adds a third dimension to MS, estimating for individual peaks their charge states z starting from z = 1 and color coding z in m/z spectra. CHARDA combines the analysis of ion signal decay rates in the time-domain data (transients) in Fourier transform (FT) MS with the interrogation of mass defects (fractional mass) of biopolymers. Being applied to individual isotopic peaks in a complex protein tandem (MS/MS) data set, CHARDA aids peptide mass spectra interpretation by facilitating charge-state deconvolution of large ionic species in crowded regions, estimating z even in the absence of an isotopic distribution (e.g., for monoisotopic mass spectra). CHARDA is fast, robust, and consistent with conventional FTMS and FTMS/MS data acquisition procedures. An effective charge-state resolution Rz ≥ 6 is obtained with the potential for further improvements.

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-α-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 µg/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.

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 for identification of diseases and discrimination of their subtypes.

Sex-Specific Silica Nanoparticle Protein Corona Compositions Exposed to Male and Female BALB/c Mice Plasmas.

As various nanoparticles (NPs) are increasingly being used in nanomedicine products for more effective and less toxic therapy and diagnosis of diseases, there is a growing need to understand their biological fate in different sexes. Herein, we report a proof-of-concept result of sex-specific protein corona compositions on the surface of silica NPs as a function of their size and porosity upon incubation with plasma proteins of female and male BALB/c mice. Our results demonstrate substantial differences between male and female protein corona profiles on the surface of silica nanoparticles. By comparing protein abundances between male and female protein coronas of mesoporous silica nanoparticles and Stöber silica nanoparticles of ∼100, 50, and 100 nm in diameter, respectively, we detected 17, 4, and 4 distinct proteins, respectively, that were found at significantly different concentrations for these constructs. These initial findings demonstrate that animal sex can influence protein corona formation on silica NPs as a function of the physicochemical properties. A more thorough consideration of the role of plasma sex would enable nanomedicine community to design and develop safer and more efficient diagnostic and therapeutic nanomedicine products for both sexes.

NCF1-dependent production of ROS protects against lupus by regulating plasmacytoid dendritic cell development and functions.

Low capacity to produce ROS because of mutations in neutrophil cytosolic factor 1 (NCF1/p47phox), a component of NADPH oxidase 2 (NOX2) complex, is strongly associated with systemic lupus erythematosus in both humans and mouse models. Here, we aimed to identify the key immune cell type(s) and cellular mechanisms driving lupus pathogenesis under the condition of NCF1-dependent ROS deficiency. Using cell-specific Cre-deleter, human NCF1-339 variant knockin, and transgenic mouse strains, we show that low ROS production in plasmacytoid dendritic cells (pDCs) exacerbated both pristane-induced lupus and a potentially new Y-linked autoimmune accelerating locus-related spontaneous model by promoting pDC accumulation in multiple organs during lupus development, accompanied by elevated IFN-α levels and expression of IFN-stimulated genes. Mechanistic studies revealed that ROS deficiency enhanced pDC generation through the AKT/mTOR pathway and CCR2-mediated migration to tissues, which together with hyperactivation of the redox-sensitive stimulator of interferon genes/IFN-α/JAK1/STAT1 cascade further augmented type I IFN responses. More importantly, by suppressing these pathways, restoration of NOX2-derived ROS specifically in pDCs protected against lupus. These discoveries explain the causative effect of dysfunctional NCF1 in lupus and demonstrate the protective role of pDC-derived ROS in disease development driven by NCF1-dependent ROS deficiency.

Haematococcus as a promising cell factory to produce recombinant pharmaceutical proteins.

The need for recombinant pharmaceutical proteins has urged scientists all over the world to search for better protein expression systems which have higher capabilities and flexibilities. Although a number of protein expression systems are now available, no system is ideal and different systems lack specific properties. Here, microalga Haematococcus is discussed as a new protein expression system which merits cheap growth medium, fast growth rate, ease of manipulation and scale-up, ease of transformation, potential of exploiting in bioreactors and ability to exert post-translational modifications to the proteins. This green single-cell plant has favorable biological and biotechnological features for production of remarkable yields of recombinant proteins with high functionality. In this review article, we highlight the favorable biotechnological characteristics of Haematococcus for lowering costs and facilitating scale-up of recombinant protein production along with its superior biological features for genetic engineering.