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Results for “top-down proteomics”

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PubMed2025

Establishing a Top-Down Proteomics Platform on a Time-of-Flight Instrument with Electron-Activated Dissociation.

Top-down proteomics is the study of intact proteins and their post-translational modifications with mass spectrometry. Historically, this field is more challenging than its bottom-up counterpart because the species are much bigger and have a larger number of possible combinations of sequences and modifications; thus, there is a great need for technological development. With improvements in instrumentation and a multiplicity of fragmentation modes available, top-down proteomics is quickly gaining in popularity with renewed attention on increasing confidence in identification and quantification. Here, we systematically evaluated the Sciex ZenoTOF 7600 system for top-down proteomics, applying standards in the field to validate the platform and further experimenting with its capabilities in electron-activated dissociation and post-translational modification site localization. The instrument demonstrated robustness in standard proteins for platform QC, as aided by zeno trapping. We were also able to apply this to histone post-translational modifications, achieving high sequence coverage that allowed PTM's site localization across protein sequences with optimized EAD fragmentation. We demonstrated the ability to analyze proteins spanning the mass range and included analysis of glycosylated proteins. This is a reference point for future top-down proteomics experiments to be conducted on the ZenoTOF 7600 system.

Proteomics↗

PubMed2025

High-Sensitivity Top-Down Proteomics Reveals Enhanced Maturation of Micropatterned Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for disease modeling, drug discovery, and precision medicine, yet their utility is often limited by their immature phenotype. One promising maturation strategy involves using micropatterned substrates that mimic native cardiomyocytes' organizational growth and stiffness. However, the maturity of this model has not fully been assessed, and there is currently no method to extract proteins from micropatterned hiPSC-CMs for top-down proteomic analysis. Herein, we present a high-sensitivity protein extraction protocol for top-down proteomic analysis of hiPSC-CMs. Through this method, we assessed the maturation of micropatterned hiPSC-CMs compared to traditional monoculture and coculture monolayers at the proteoform level. We found that micropatterned hiPSC-CMs display molecular signatures of cardiomyocyte maturation including increased expression of ventricular myosin light chain isoforms, reduced expression of the fetal troponin T isoform, and decreased phosphorylation of alpha-tropomyosin. This high-sensitivity approach enables robust top-down proteomics from limited, heterogeneous cell populations and identifies the micropattern hiPSC-CM as a more adult-like CM model, broadening the utility of structured culture systems for cardiac disease modeling and translational research. Source data for this manuscript are available via MassIVE repository at massive.ucsd.edu with identifier: MSV000097864.

Myocytes, Cardiac↗

PubMed2024

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.

Proteomics↗

PubMed2024

Top-Down Proteomics Identifies Plasma Proteoform Signatures of Liver Cirrhosis Progression.

Cirrhosis, advanced liver disease, affects 2 to 5 million Americans. While most patients have compensated cirrhosis and may be fairly asymptomatic, many decompensate and experience life-threatening complications such as gastrointestinal bleeding, confusion (hepatic encephalopathy), and ascites, reducing life expectancy from 12 to less than 2 years. Among patients with compensated cirrhosis, identifying patients at high risk of decompensation is critical to optimize care and reduce morbidity and mortality. Therefore, it is important to preferentially direct them towards specialty care which cannot be provided to all patients with cirrhosis. We used discovery top-down proteomics to identify differentially expressed proteoforms (DEPs) in the plasma of patients with progressive stages of liver cirrhosis with the ultimate goal to identify candidate biomarkers of disease progression. In this pilot study, we identified 209 DEPs across three stages of cirrhosis (compensated, compensated with portal hypertension, and decompensated), of which 115 derived from proteins enriched in the liver at a transcriptional level and discriminated the three stages of cirrhosis. Enrichment analyses demonstrated DEPs are involved in several metabolic and immunological processes known to be impacted by cirrhosis progression. We have preliminarily defined the plasma proteoform signatures of cirrhosis patients, setting the stage for ongoing discovery and validation of biomarkers for early diagnosis, risk stratification, and disease monitoring.

Humans↗

PubMed2025

Mass spectrometry-based top-down proteomics for proteoform profiling of protein coronas.

The protein corona is a layer of biomolecules-primarily proteins-that adsorbs to nanoparticle (NP) surfaces in biological fluids. If the purpose of the NP is therapeutic, this can have a profound effect on its biological activity and function in vivo. Protein corona formation can also be exploited for diagnostic purposes and to differentially enrich proteins for biomarker discovery. For all of these applications, it is useful to determine which proteins, and which specific proteoforms, bind to different types of NP. The traditional mass spectrometry (MS)-based bottom-up proteomics does not accurately identify specific proteoforms within the protein corona. This limitation impedes the nanomedicine field's ability to precisely predict the biological fate and pharmacokinetics of nanomedicines and their effectiveness in early-stage biomarker discovery and disease detection because many different proteoforms of the same gene could exist in the corona, and they have divergent biological functions. Here, we describe how to use capillary zone electrophoresis (CZE)-MS-based top-down proteomics to characterize the proteoform landscape of the protein corona. Our procedures detail the recovery of intact proteoforms from NP surfaces by using detergent-assisted proteoform elution and the measurement of these proteoforms by using CZE-tandem MS (MS/MS) and CZE-high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS/MS. The entire workflow is completed within 3-4 d. Using this protocol, hundreds of proteoforms from the protein corona of polystyrene NPs can be identified. Distinct protein corona proteoform profiles were observed from NPs with different physicochemical properties. The addition of FAIMS is beneficial for more in-depth proteoform characterization.

Proteomics↗

PubMed2026

A Draft Map of E. coli Proteoforms.

Top-down proteomics (TDP) enables direct characterization of intact proteoforms, providing protein-level insights into molecular diversity arising from post-translational modifications and sequence variations. Despite this advantage, proteome coverage in TDP remains limited relative to bottom-up proteomics (BUP). To expand coverage, we developed an integrated multidimensional approach combining sequential protein extraction, size-exclusion chromatography (SEC) fractionation, and capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS. This approach identified 743 proteoform families and 10,613 proteoforms from E. coli cells through hundreds of MS runs. By incorporating previous E. coli TDP data sets from our group, we identified 14,932 proteoforms from 985 proteoform families, covering 43% of the E. coli proteome. The data represent the highest proteome coverage of cells by MS-based TDP, creating a draft map of E. coli proteoforms. The results offer strong evidence that MS-based TDP can reach high proteome coverage.

Escherichia coli↗

PubMed2025

Uncovering hidden protein modifications with native top-down mass spectrometry.

Protein modifications drive dynamic cellular processes by modulating biomolecular interactions, yet capturing these modifications within their native structural context remains a significant challenge. Native top-down mass spectrometry promises to preserve the critical link between modifications and interactions. However, current methods often fail to detect uncharacterized or low-abundance modifications, limiting insights into proteoform diversity. To address this gap, we introduce precise and accurate Identification Of Native proteoforms (precisION), an interactive end-to-end software package that leverages a robust, data-driven fragment-level open search to detect, localize and quantify 'hidden' modifications within intact protein complexes. Applying precisION to four therapeutically relevant targets-PDE6, ACE2, osteopontin (SPP1) and a GABA transporter (GAT1)-we discover undocumented phosphorylation, glycosylation and lipidation, and resolve previously uninterpretable density in an electron cryo-microscopy map of GAT1. As an open-source software package, precisION offers an intuitive means for interpreting complex protein fragmentation data. This tool will empower the community to unlock the potential of native top-down mass spectrometry, advancing integrative structural biology, molecular pathology and drug development.

Mass Spectrometry↗