The TET3 inflammasome senses unique long HSV-1 proteins for virus particle budding from the nucleus.

Inflammasomes play important roles in resisting infections caused by various pathogens. HSV-1 is a highly contagious virus among humans. The process by which HSV-1 particles bud from the nucleus is unique to herpes viruses, but the specific mechanism is still unclear. Here, we screened genes involved in HSV-1 replication. We found that TET3 plays an essential role in HSV-1 infection. TET3 recognizes the UL proteins of HSV-1 and, upon activation, can directly bind to caspase-1 to activate an ASC-independent inflammasome in the nucleus. The subsequent cleavage of GSDMD in the nucleus is crucial for the budding of HSV-1 particles from the nucleus. Inhibiting the perforation ability of GSDMD on the nuclear membrane can significantly reduce the maturation and spread of HSV-1. Our results may provide a new approach for the treatment of HSV-1 in the future.

Deciphering nanoparticle protein coronas by capillary isoelectric focusing-mass spectrometry-based top-down proteomics.

The nanoparticle (NP) protein corona significantly influences the outcome of nanomedicine. We present the first example of top-down proteomics (TDP) measurement of the protein corona using capillary isoelectric focusing-mass spectrometry, identifying seventy proteoforms of 16 cancer-related genes. This technique has the potential to revolutionize our understanding of the protein corona and advance nanomedicine.

The bifunctional transcription factor NAC32 modulates nickel toxicity responses through repression of root-nickel compartmentalization and activation of auxin biosynthesis.

Nickel (Ni) is an important micronutrient, but excess Ni is toxic to many plant species. Currently, relatively little is known about the genetic basis of the plant responses to Ni toxicity. Here, we demonstrate that NAC32 transcription factor functions as a core genetic hub to regulate the Ni toxicity responses in Arabidopsis. NAC32 negatively regulates root-Ni concentration through the IREG2 (IRON REGULATED2) encoding a transporter. NAC32 also induces local auxin biosynthesis in the root-apex transition zone by upregulating YUCCA 7 (YUC7)/8/9 expression, which results in a local enhancement of auxin signaling in root tips, especially under Ni toxicity, thereby impaired primary root growth. By analyses of various combinations of nac32 and ireg2 mutants, as well as nac32 and yuc7/8/9 triple mutants, including high-order quadruple mutant, we demonstrated that NAC32 negatively regulates Ni stress tolerance by acting upstream of IREG2 and YUC7/8/9 to modulate their function in Ni toxicity responses. ChIPqPCR, EMSA (electrophoretic mobility shift assay) and transient dual-LUC reporter assays showed that NAC32 transcriptionally represses IREG2 expression but activates YUC7/8/9 expression by directly binding to their promoters. Our work demonstrates that NAC32 coordinates Ni compartmentation and developmental plasticity in roots, providing a conceptual framework for understanding Ni toxicity responses in plants.

Pilot Study for Deciphering Post-Translational Modifications and Proteoforms of Tau Protein by Capillary Electrophoresis-Mass Spectrometry.

Abnormal accumulation of tau protein in the brain is one pathological hallmark of Alzheimer's disease (AD). Many tau protein post-translational modifications (PTMs) are associated with the development of AD, such as phosphorylation, acetylation, and methylation. Therefore, a complete picture of the PTM landscape of tau is critical for understanding the molecular mechanisms of AD progression. Here, we offered a pilot study of combining two complementary analytical techniques, capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of recombinant human tau-0N3R. We identified 50 phosphorylation sites of tau-0N3R in total, which is about 25% higher than that from RPLC-MS/MS alone. CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic mobility helped improve the confidence of the identified modified peptides. We developed a highly efficient capillary isoelectric focusing (cIEF)-MS technique to offer a bird's-eye view of tau-0N3R proteoforms, with 11 putative tau-0N3R proteoforms carrying up to nine phosphorylation sites and lower pI values from more phosphorylated proteoforms detected. Interestingly, under native-like cIEF-MS conditions, we observed three putative tau-0N3R dimers carrying phosphate groups. The findings demonstrate that CE-MS is a valuable analytical technique for the characterization of tau PTMs, proteoforms, and even oligomerization.

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.

A simple and efficient approach for preparing cationic coating with tunable electroosmotic flow for capillary zone electrophoresis-mass spectrometry-based top-down proteomics.

BACKGROUND: Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has become a valuable analytical technique in top-down proteomics (TDP). CZE-MS/MS-based TDP typically employs separation capillaries with neutral coatings (i.e., linear polyacrylamide, LPA). However, issues related to separation resolution and reproducibility remain with the LPA-coated capillaries due to the unavoidable non-specific protein adsorption onto the capillary wall. Cationic coatings can be critical alternatives to LPA coating for CZE-MS/MS-based TDP due to the electrostatic repulsion between the positively charged capillary inner wall and proteoform molecules in the acidic separation buffer. Unfortunately, there are only very few studies using cationic coating-based CZE-MS/MS for TDP studies. RESULTS: In this work, we aimed to develop a simple and efficient approach for preparing separation capillaries with a cationic coating, i.e., poly (acrylamide-co-(3-acrylamidopropyl) trimethylammonium chloride [PAMAPTAC]) for CZE-MS/MS-based TDP. The PAMAPTAC coating-based CZE-MS produced significantly better separation resolution of proteoforms compared to the traditionally used LPA-coated approach. It achieved reproducible separation and measurement of a simple proteoform mixture and a complex proteome sample (i.e., a yeast cell lysate) regarding migration time, proteoform intensity, and the number of proteoform identifications. The PAMAPTAC coating-based CZE-MS enabled the detection of large proteoforms (≥30 kDa) from the yeast cell lysate reproducibly without any size-based prefractionation. Interestingly, the mobility of proteoforms using the PAMAPTAC coating can be predicted accurately using a simple semi-empirical model. SIGNIFICANCE: The results render the PAMAPTAC coating as a valuable alternative to the LPA coating to advance CZE-MS-based TDP towards high-resolution separation and highly reproducible measurement of proteoforms in complex samples.

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 the 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 the nanomedicine community to partly identify the biological identity of NPs. Such an approach, however, fails to pinpoint the specific proteoforms─distinct molecular variants of proteins in the protein corona. The proteoform-level information could potentially advance the 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 characterizing proteoforms in the protein corona. Our TDP approach has successfully identified about 900 proteoforms in the protein corona of polystyrene NPs, ranging from 2 to 70 kDa, revealing proteoforms of 48 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 advanced approach to characterize NP protein coronas, deepening our understanding of NPs' biological identities. We, therefore, propose using both TDP and BUP strategies to obtain more comprehensive information about the protein corona, which, in turn, can further enhance the diagnostic and therapeutic efficacy of nanomedicine technologies.

PNLDC1 catalysis and postnatal germline function are required for piRNA trimming, LINE1 silencing, and spermatogenesis in mice.

PIWI-interacting RNAs (piRNAs) play critical and conserved roles in transposon silencing and gene regulation in the animal germline. Three distinct piRNA populations are present during mouse spermatogenesis: fetal piRNAs in fetal/perinatal testes, pre-pachytene and pachytene piRNAs in postnatal testes. PNLDC1 is required for piRNA 3' end maturation in multiple species. However, whether PNLDC1 is the bona fide piRNA trimmer and the physiological role of 3' trimming of different piRNA populations in spermatogenesis in mammals remain unclear. Here, by inactivating Pnldc1 exonuclease activity in vitro and in mice, we reveal that the PNLDC1 trimmer activity is essential for spermatogenesis and male fertility. PNLDC1 catalytic activity is required for both fetal and postnatal piRNA 3' end trimming. Despite this, postnatal piRNA trimming but not fetal piRNA trimming is critical for LINE1 transposon silencing. Furthermore, conditional inactivation of Pnldc1 in postnatal germ cells causes LINE1 transposon de-repression and spermatogenic arrest in mice, indicating that germline-specific postnatal piRNA trimming is essential for transposon silencing and germ cell development. Our findings highlight the germ cell-intrinsic role of PNLDC1 and piRNA trimming in mammals to safeguard the germline genome and promote fertility.

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry.

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

A pilot study for deciphering post-translational modifications and proteoforms of tau protein by capillary electrophoresis-mass spectrometry.

Abnormal accumulation of tau proteins is one pathological hallmark of Alzheimer□s disease (AD). Many tau protein post-translational modifications (PTMs) are associated with the development of AD, such as phosphorylation, acetylation, and methylation. Therefore, a complete picture of PTM landscape of tau is critical for understanding the molecular mechanisms of AD progression. Here, we offered a pilot study of combining two complementary analytical techniques, capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of recombinant human tau-0N3R. We identified 53 phosphorylation sites of tau-0N3R in total, which is about 30% higher than that from RPLC-MS/MS alone. CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic mobility helped improve the confidence of the identified modified peptides. We developed a highly efficient capillary isoelectric focusing (cIEF)-MS technique to offer a bird's-eye view of tau-0N3R proteoforms, with 11 putative tau-0N3R proteoforms carrying up to nine phosphorylation sites and lower pI values from more phosphorylated proteoforms detected. Interestingly, under a native-like cIEF-MS condition, we observed three putative tau-0N3R dimers carrying phosphate groups. The findings demonstrate that CE-MS is a valuable analytical technique for the characterization of tau PTMs, proteoforms, and even oligomerization.

Mass spectrometry-intensive top-down proteomics: an update on technology advancements and biomedical applications.

Proteoforms are all forms of protein molecules from the same gene because of variations at the DNA, RNA, and protein levels, e.g., alternative splicing and post-translational modifications (PTMs). Delineation of proteins in a proteoform-specific manner is crucial for understanding their biological functions. Mass spectrometry (MS)-intensive top-down proteomics (TDP) is promising for comprehensively characterizing intact proteoforms in complex biological systems. It has achieved substantial progress in technological development, including sample preparation, proteoform separations, MS instrumentation, and bioinformatics tools. In a single TDP study, thousands of proteoforms can be identified and quantified from a cell lysate. It has also been applied to various biomedical research to better our understanding of protein function in regulating cellular processes and to discover novel proteoform biomarkers of diseases for early diagnosis and therapeutic development. This review covers the most recent technological development and biomedical applications of MS-intensive TDP.

SNX14 inhibits autophagy via the PI3K/AKT/mTOR signaling cascade in breast cancer cells.

BACKGROUND: Sorting nexin 14 (SNX14) is a member of the sorting junction protein family. Its specific roles in cancer development remain unclear. Therefore, in this study, we aimed to determine the effects and underlying mechanisms of SNX14 on autophagy of breast cancer cells to aid in the therapeutic treatment of breast cancer. METHODS: In this study, we performed in vitro experiments to determine the effect of SNX14 on breast cancer cell growth. Moreover, we used an MCF7 breast cancer tumor-bearing mouse model to confirm the effect of SNX14 on tumor cell growth in vivo. We also performed western blotting and quantitative polymerase chain reaction to identify the mechanism by which SNX14 affects breast cancer MCF7 cells. RESULTS: We found that SNX14 regulated the onset and progression of breast cancer by promoting the proliferation and inhibiting the autophagy of MCF7 breast cancer cells. In vivo experiments further confirmed that SNX14 knockdown inhibited the tumorigenicity and inhibited the growth of tumor cells in tumor tissues of nude mice. In addition, western blotting analysis revealed that SNX14 modulate the autophagy of MCF7 breast cancer cells via the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin kinase signaling pathway. CONCLUSION: Our findings indicate that SNX14 is an essential tumor-promoting factor in the development of breast cancer.

Dynamic bond crosslinked maca polysaccharide hydrogels with reactive oxygen species scavenging and antibacterial effects on infected wound healing.

In this study, a polysaccharide fragment with antioxidant and reactive oxygen species (ROS) scavenging activities was extracted from Maca (Lepidium meyenii Walp.) and subjected to structural analyses. The fragment, characterized by the α-D-Glcp-(1 â†’ terminal group of the main chain linked to the →4)-Glcp-(1 â†’ end unit through an O-6 bond and the O-3 bond of 1-3-4Glcp, was modified by introducing dialdehyde structures on its glucose units. It was then crosslinked with N-carboxymethyl chitosan via the Schiff base reaction to create a multifunctional hydrogel with antibacterial and ROS scavenging properties. Polyvinyl alcohol was incorporated to form a double crosslinked gel network, and the addition of silver nanoparticles enhanced its antibacterial efficacy. This gel system can scavenge excess ROS, mitigate wound inflammation, eradicate harmful bacteria, and aid in the restoration of skin microecology. The multifunctional maca polysaccharide hydrogel shows significant potential as a medical dressing for the treatment of infected wounds.

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry.

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E . coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

Quantitative proteomics reveals the dynamic proteome landscape of zebrafish embryos during the maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is central to early embryogenesis. However, its underlying molecular mechanisms are still not well described. Here, we revealed the expression dynamics of 5,000 proteins across four stages of zebrafish embryos during MZT, representing one of the most systematic surveys of proteome landscape of the zebrafish embryos during MZT. Nearly 700 proteins were differentially expressed and were divided into six clusters according to their expression patterns. The proteome expression profiles accurately reflect the main events that happen during the MZT, i.e., zygotic genome activation (ZGA), clearance of maternal mRNAs, and initiation of cellular differentiation and organogenesis. MZT is modulated by many proteins at multiple levels in a collaborative fashion, i.e., transcription factors, histones, histone-modifying enzymes, RNA helicases, and P-body proteins. Significant discrepancies were discovered between zebrafish proteome and transcriptome profiles during the MZT. The proteome dynamics database will be a valuable resource for bettering our understanding of MZT.

TopDIA: A Software Tool for Top-Down Data-Independent Acquisition Proteomics.

Top-down mass spectrometry is widely used for proteoform identification, characterization, and quantification owing to its ability to analyze intact proteoforms. In the last decade, top-down proteomics has been dominated by top-down data-dependent acquisition mass spectrometry (TD-DDA-MS), and top-down data-independent acquisition mass spectrometry (TD-DIA-MS) has not been well studied. While TD-DIA-MS produces complex multiplexed tandem mass spectrometry (MS/MS) spectra, which are challenging to confidently identify, it selects more precursor ions for MS/MS analysis and has the potential to increase proteoform identifications compared with TD-DDA-MS. Here we present TopDIA, the first software tool for proteoform identification by TD-DIA-MS. It generates demultiplexed pseudo MS/MS spectra from TD-DIA-MS data and then searches the pseudo MS/MS spectra against a protein sequence database for proteoform identification. We compared the performance of TD-DDA-MS and TD-DIA-MS using Escherichia coli K-12 MG1655 cells and demonstrated that TD-DIA-MS with TopDIA increased proteoform and protein identifications compared with TD-DDA-MS.

Bounding the Amount of Entanglement from Witness Operators.

We present an approach to estimate the operational distinguishability between an entangled state and any separable state directly from measuring an entanglement witness. We show that this estimation also implies bounds on a variety of other well-known entanglement quantifiers. This approach for entanglement estimation is then extended to both the measurement-device-independent scenario and the fully device-independent scenario, where we obtain nontrivial but suboptimal bounds. The procedure requires no numerical optimization and is easy to compute. It offers ways for experimenters to not only detect, but also quantify, entanglement from the standard entanglement witness procedure.

Microfluidic impedance cytometry with flat-end cylindrical electrodes for accurate and fast analysis of marine microalgae.

Marine microalgae play an increasingly significant role in addressing the issues of environmental monitoring and disease treatment, making the analysis of marine microalgae at the single-cell level an essential technique. For this, we put forward accurate and fast microfluidic impedance cytometry to analyze microalgal cells by assembling two cylindrical electrodes and microchannels to form a three-dimensional detection zone. Firstly, we established a mathematical model of microalgal cell detection based on Maxwell's mixture theory and numerically investigated the effects of the electrode gap, microalgal positions, and ion concentrations of the solution on detection to optimize detection conditions. Secondly, 80 µm stainless steel wires were used to construct flat-ended cylindrical electrodes and were then inserted into two collinear channels fabricated using standard photolithography techniques to form a spatially uniform electric field to promote the detection throughput and sensitivity. Thirdly, based on the validation of this method, we measured the impedance of living Euglena and Haematococcus pluvialis to study parametric influences, including ion concentration, cell density and electrode gap. The throughput of this method was also investigated, which reached 1800 cells per s in the detection of Haematococcus pluvialis. Fourthly, we analyzed live and dead Euglena to prove the ability of this method to detect the physiological status of cells and obtained impedances of 124.3 Ω and 31.0 Ω with proportions of 15.9% and 84.1%, respectively. Finally, this method was engineered for the analysis of marine microalgae, measuring living Euglena with an impedance of 159.61 Ω accounting for 3.9%, dead Euglena with an impedance of 36.43 Ω accounting for 10.1% and Oocystis sp. with an impedance of 55.00 Ω accounting for about 81.0%. This method could provide a reliable tool to analyze marine microalgae for monitoring the marine environment and treatment of diseases owing to its outstanding advantages of low cost, high throughput and high corrosion resistance.

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.