A key limitation of top-down proteomics is reliance on averagine-based deconvolution to estimate monoisotopic masses, which introduces systematic errors when isotope envelopes are distorted. We present a framework that bypasses averagine by operating directly in natural log-transformed m/z space, where charge-state spacing is mass-invariant and provides an intrinsic reference for internal calibration on both FT-ICR and Orbitrap analyzers. Isotopologue pairing in this domain supports de novo sequencing and discriminates near-isobaric residues. By shifting the paradigm from monoisotopic mass estimation to connectivity-driven inference, the approach offers resilience against distorted isotope envelopes and unknown PTMs, establishing a database-independent strategy for discovery-oriented proteoform characterization without known calibrants.

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been documented as a useful tool for top-down proteomics (TDP). However, CZE-MS/MS-based TDP typically has limited backbone cleavage coverage for identified proteoforms due to the use of traditional collision-based fragmentation methods (i.e., higher-energy collisional dissociation, HCD). Here, for the first time, we coupled CZE to an Orbitrap Ascend Tribrid mass spectrometer to investigate the performance of collision-, electron-, and photon-based fragmentation methods and their combinations for boosting the backbone cleavage coverage of proteoforms during the electrophoretic time scale using a standard protein mixture covering a mass range of about 10-70 kDa. CZE-MS achieved reproducible measurement of six proteins including three insulin-like growth factor (IGF) proteoforms with different modifications. Systematic investigations of HCD, electron-transfer dissociation (ETD), electron-transfer/HCD (EThcD), and ultraviolet photodissociation (UVPD) during CZE-MS/MS analysis revealed distinct yet complementary fragmentation characteristics. ETD, EThcD, and UVPD, in general, provided higher backbone cleavage coverage than HCD. The integration of HCD, ETD, EThcD, and UVPD data offered 67 and 98% sequence coverage for carbonic anhydrase (a 30 kDa protein) and thioredoxin (a 12 kDa protein), which is 158 and 100% higher than that produced by HCD alone. Adding internal fragments further boosted the backbone cleavage coverage substantially, for example, from 67 to 94% for 30 kDa carbonic anhydrase and from 21 to 82% for 50 kDa protein AG. The results demonstrate the capability of CZE-MS/MS with the integration of various fragmentation techniques for comprehensive characterization of proteoforms in a wide mass range.

Top-down proteomics (TDP) enables the characterization of histone proteoforms. However, the extensive post-translational modifications and high sequence homology of these proteins pose significant separation challenges. Here, we compare C4 and diphenyl reversed-phase columns for top-down analysis of histone proteoforms. The TDP-based methods were developed using a mixture of six intact protein standards (~9-68 kDa) and applied to a cellular histone extract. While the C4 stationary phase favored the retention of more hydrophobic proteoforms, the diphenyl phase enhanced the separation of more polar species. In total, 55 histone proteoforms were identified, including 14 unique to C4 and 19 unique to diphenyl. These results demonstrate the orthogonal selectivity of the two stationary phases and highlight the importance of column chemistry in improving proteoform resolution in TDP workflows.

ABSTRACTCapillary zone electrophoresis–mass spectrometry (CZE‐MS) has become a powerful tool for top‐down proteomics (TDP). Capillaries are commonly coated with neutral or cationic polymers to reduce protein adsorption in CZE‐MS. We recently introduced a cationic polymer coating [poly(acrylamide‐co‐(3‐acrylamidopropyl) trimethylammonium chloride (PAMAPTAC)] to CZE‐MS–based TDP. Here, we further improved the coating procedure to enhance the ease and repeatability of the cationic coating preparation by employing 2,2′‐azobisisobutyronitrile (AIBN) as the radical initiator and methanol as the solvent. The new coating method not only simplifies the coating process but is also user‐friendly. Different laboratory members were able to reproduce this type of cationic coating with consistent CZE‐MS performance. We evaluated the performance of CZE‐MS with the cationic coating in analyzing various complex biological samples (nanoparticle protein corona, histone, and an Escherichia coli cell lysate) and assessed its long‐term reproducibility with an E. coli cell lysate for 35 runs (over 40 h) of continuous measurement. The CZE‐MS system produced reproducible measurements for all the samples regarding separation profiles, proteoform migration time (MT), and proteoform intensity. For example, the system enabled the detection of large proteoforms (26–35 kDa) from an E. coli sample with consistent MT, relative standard deviations (RSDs) <7% without MT correction and 2.1% after MT alignment. We expect that the APTAC coating will further advance CZE‐MS–based TDP for broad and large‐scale applications.

Mass spectrometry (MS)‐based top–down proteomics (TDP) has emerged as a powerful tool for characterizing proteoforms to advance both fundamental and translational research. TDP requires high‐efficiency liquid‐phase separation, high‐resolution MS, and tandem MS. Capillary zone electrophoresis (CZE)‐MS has been proposed as a promising analytical technique for protein analysis decades ago because of its unique and valuable features, including high separation efficiency and high detection sensitivity. However, CZE‐MS has not been widely adopted by the proteomics community, mainly due to concerns with its robustness and reproducibility. Here, we hypothesized that CZE‐MS is sufficiently robust and reproducible for broad adoption due to the continued efforts of the community over the last three decades. In this work, for the first time, research teams from around the world validated the robustness, repeatability, and reproducibility of CZE‐MS for TDP in both simple and complex model proteoform mixtures employing a full spectrum of commercially available capillary electrophoresis (CE)‐MS interfaces, instrumentation, and compared CZE‐MS performance with state‐of‐the‐art liquid chromatography (LC)‐MS methods. This study offers the research community an informative resource of ready‐to‐use experimental CE‐MS techniques and a better understanding of the CZE‐MS approach and its potential in TDP, accelerating the broad adoption of CZE‐MS in proteoform research.

Cellular senescence is a stable state of cell-cycle arrest characterized by extensive remodeling of the secretome, known as the senescence-associated secretory phenotype (SASP). The SASP profoundly influences tissue microenvironments and contributes to chronic inflammation and age-related diseases. While previous studies have characterized the SASP using bottom-up proteomics, intact proteoforms' diversity and structural complexity remain poorly understood. In this study, we apply quantitative top-down mass spectrometry to profile the intact proteoform composition of the SASP in senescent human fibroblasts, alongside quiescent and proliferating controls. This approach enables direct identification of intact proteoforms with post-translational modifications (PTMs), sequence variants, and isoforms, offering deep insight into the proteomic landscape of senescence. We identify a rich repertoire of previously uncharacterized proteoforms, including variants of HMGA2 with N-terminal acetylation and multiple phosphorylation states (di-, tri-, and tetra-phosphorylated), implicating them as potential senescence biomarkers. Our findings underscore the functional complexity of the SASP and the value of proteoform-level resolution in understanding cellular senescence. This work establishes a robust top-down proteomics strategy for SASP analysis and highlights novel molecular targets for therapeutic strategies aimed at mitigating age-related pathologies.

Hypertrophic cardiomyopathy (HCM) has traditionally been regarded as a disease of the sarcomere; however, it is in the midst of a paradigm shift with growing recognition of contributions beyond the sarcomere to the heterogeneity of HCM phenotypes. Innovative approaches are essential to uncover novel determinants and mechanisms underlying this heterogeneity. Top-down proteomics has emerged as a powerful method for analysis of proteoforms-the myriad protein products arising from genetic variants, posttranslational modifications, and splicing isoforms from a single gene-offering a more precise lens to understand the disease heterogeneity in HCM. Yet, how proteoforms are altered on a global scale in HCM has not been investigated. Global top-down proteomics was performed on myocardial samples from patients with advanced obstructive HCM and nonfailing controls. Specifically, serial protein extraction enabled by the photocleavable surfactant, 4-hexylphenylazosulfonate (Azo), was utilized to solubilize diverse categories of proteins from minimal tissue, including membrane proteins. Subsequently, high-sensitivity top-down mass spectrometry was used to detect and quantify proteoforms across various cellular compartments. Using this global top-down proteomics approach, we have detected ≈2000 proteoforms across disparate cellular compartments, including the sarcoplasmic reticulum, cytoskeleton, mitochondria, and nucleus, in advanced obstructive HCM tissues. Quantitative analysis uncovered significant alterations not only in sarcomeric but also cytoskeletal, mitochondrial, nucleosome, and sarcoplasmic reticulum proteoforms in HCM as compared with nonfailing controls. Notably, we have discovered a significant proteoform crosstalk among the sarcomere, sarcoplasmic reticulum, and cytoskeleton. Moreover, we have identified a previously unrecognized decrease in succinylated mitochondrial proteoforms as a critical feature of the advanced obstructive HCM proteoform landscape, alongside a marked reduction in acetylation of nucleosome proteins. This study represents the most comprehensive analysis of the proteoform landscape in HCM to date, uncovering pathways beyond the sarcomere that may contribute to HCM pathophysiology and identifying potential targets for development of therapeutic interventions.

Mass spectrometry-based proteomics is a versatile technique that facilitates the study of microproteins as biomarkers and potential translational applications by utilizing liquid chromatography-trapped ion mobility-tandem mass spectrometry (LC-TIMS-MS/MS) and matrix assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Importantly, MS has been shown to be compatible with complex biological samples (i.e., mammalian biofluids and patient samples). Here we describe a workflow combining: (1) top-down proteomics for analysis of intact microproteins via MALDI-TOF MS and (2) bottom-up proteomics for analysis of enzymatically digested microproteins via LC-TIMS-MS/MS to annotate putative microproteins of interest from a complex biological sample: patient-derived tampons.

Properties, Origin, and Consistency of Truncated Proteoforms Across Top-Down Proteomic Studies

ABSTRACTProteoforms, the endogenous forms of proteins that take into account all sources of variation (genetic as well as co‐ and post‐translational), may be more relevant for understanding complex biological mechanisms, including disease phenotypes, than generically defined “protein families”. Mass spectrometry‐based top‐down proteomics often utilizes electrophoretic techniques that rely on sodium dodecyl sulfate (SDS) to fractionate proteoforms prior to liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis. Methanol‐chloroform‐water (MCW) precipitation is commonly utilized for SDS removal due to its efficacy and low cost, yet it may lead to poor recovery of smaller proteoforms. As a technical contribution to top‐down proteomics, four commercial SDS clean‐up alternatives were benchmarked against MCW. Results indicate that MCW yields fewer proteoform identifications, particularly among small and acidic proteoforms. The analysis of post‐translational modifications identified using the different clean‐up methods indicates increased prevalence of methylation modifications post–MCW clean‐up. Among the commercial kits, DetergentOUT and HiPPR achieved SDS removal comparable to MCW but at a higher cost. For studies sensitive to loss of low molecular weight or acidic proteoforms, these kits may offer an advantage. Alternatively, at a lower cost, Minute SDS provides sufficient SDS removal and broader proteome coverage.SummaryThis study aims to support the growing top‐down proteomics (TDP) community by addressing a practical challenge in sample preparation: efficient SDS removal following molecular weight‐based fractionation. As TDP gains traction for its ability to directly characterize proteoforms and their post‐translational modifications (PTMs), accessible and reliable workflows become increasingly important. Detergent removal is a key step, as SDS, though essential for protein solubilization and separation, interferes with mass spectrometry. Traditional methods like methanol‐chloroform‐water (MCW) precipitation can be labor‐intensive and may lead to loss of small proteoforms. In this work, we compare four commercially available SDS clean‐up kits to MCW based on SDS removal efficiency, proteoform recovery, PTM retention, and ease of use. Our findings provide practical guidance regarding each method's trade‐offs, aiming to inform more streamlined, reproducible TDP workflows. By benchmarking these kits, we contribute to ongoing efforts to democratize TDP and enhance its utility in biological and clinical research.The golden standard for SDS removal is methanol‐chloroform‐water (MCW) precipitation, which is labor‐intensive and, as demonstrated by both earlier reports and the present work, may lead to loss of small proteoforms.This study benchmarks four commercially available SDS clean‐up kits against MCW, considering SDS removal efficiency, proteoform recovery, retention of post‐translationally modified proteoforms, and ease of use.Resin‐based clean‐up methods, DetergentOUT and HiPPR, were highly effective in SDS removal, and comparable to MCW clean‐up.Minute SDS and SurfactAway kits, on the other hand, allowed the identification of a slightly higher number of proteoforms.

A central challenge in top‐down proteomics (TDP) is the characterization of large proteoforms (>70 kDa) due to their high spectral complexity in mass spectrometers. Here, we advance individual ion mass spectrometry (I2MS) for intact mass and fragmentation analysis of β‐ and α‐catenins (85–110 kDa), key components of adherens junctions. Using denatured I2MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on β‐catenin (β‐cat) and 7 on α‐catenin (α‐cat), with site‐specific changes corresponding to actomyosin contractility. Notably, phosphorylation at α‐cat S641 was constitutive, while other sites in the P‐linker and actin‐binding domains, as well as β‐cat S675 and S552, were sensitive to actomyosin perturbation. Application of I2MS for fragment ion detection (I2MS2) also enabled 25%–30% sequence coverage for these exceptionally large proteoforms, compared to <1% using conventional methods for top‐down mass spectrometry (MS). Our results are consistent with a “catenin phospho‑code” model, wherein combinatorial phosphorylation patterns reflect and potentially modulate the mechanotransductive environment at cell–cell adhesions. This work establishes top‐down I2MS as a viable approach for probing complex post‐translational modification (PTM) landscapes in high‐mass proteins and highlights proteoforms as functional units in cellular regulation.

Abstract A central challenge in top‐down proteomics (TDP) is the characterization of large proteoforms (&gt;70 kDa) due to their high spectral complexity in mass spectrometers. Here, we advance individual ion mass spectrometry (I 2 MS) for intact mass and fragmentation analysis of β‐ and α‐catenins (85–110 kDa), key components of adherens junctions. Using denatured I 2 MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on β‐catenin (β‐cat) and 7 on α‐catenin (α‐cat), with site‐specific changes corresponding to actomyosin contractility. Notably, phosphorylation at α‐cat S641 was constitutive, while other sites in the P‐linker and actin‐binding domains, as well as β‐cat S675 and S552, were sensitive to actomyosin perturbation. Application of I 2 MS for fragment ion detection (I 2 MS 2 ) also enabled 25%–30% sequence coverage for these exceptionally large proteoforms, compared to &lt;1% using conventional methods for top‐down mass spectrometry (MS). Our results are consistent with a “catenin phospho‑code” model, wherein combinatorial phosphorylation patterns reflect and potentially modulate the mechanotransductive environment at cell–cell adhesions. This work establishes top‐down I 2 MS as a viable approach for probing complex post‐translational modification (PTM) landscapes in high‐mass proteins and highlights proteoforms as functional units in cellular regulation.

Mass spectrometry (MS)-based proteomics methods, including protein footprinting methods such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) and hydroxyl radical footprinting (HRF), can give unique insight into protein structure and interactions. These methods primarily utilize bottom-up proteomics techniques that require the digestion of intact proteins into small peptides before MS analysis. This digestion can obscure structural information relevant to the function of the intact proteoforms. Here, we have developed a novel top-down footprinting method, Methionine Oxidation Footprinting in Intact Proteins (MOFIP), to probe solvent accessibility in intact proteoforms. For MOFIP, natively folded protein lysates are incubated with and without hydrogen peroxide (H2O2) to evaluate solvent accessibility of methionine residues. Top-down proteomics analysis allows the characterization of the solvent accessibility of each methionine residue within intact proteins to obtain structural information. Here, intact proteins in Escherichia coli (E. coli) lysate were used to evaluate the feasibility of complex biological sample analysis using the MOFIP platform. In total, we characterized 69 quantifiable proteoforms that contained at least one methionine residue suitable for methionine footprinting. We evaluated the oxidation state of individual methionine residues within each proteoform upon exposure to H2O2 to determine accessibility to solvent. Upon incubation with H2O2, solvent-accessible methionine residues were fully oxidized, and solvent-inaccessible residues remained unoxidized. Moreover, partially accessible residues showed incomplete oxidation, which may suggest more than one conformation existing in the native cell lysate (e.g., binding and free states). Overall, our novel MOFIP approach successfully characterized the solvent accessibility of methionine residues in intact proteins within complex samples, providing insights into proteoform structure that is difficult to obtain using bottom-up proteomics. SUMMARY: We introduce methionine oxidative footprinting for intact proteins (MOFIP), a top-down proteomics technique that enables high-throughput analysis of methionine solvent accessibility at the intact proteoform level. MOFIP has been benchmarked using an E. coli model system, and methionine accessibility was examined in 69 intact proteoforms. Determining methionine accessibility in intact proteins provides structural insights into proteoforms that are difficult to obtain using bottom-up proteomics methods.

Ultrasensitive top-down proteomics techniques provide valuable insights into PTM-regulated cellular functions in mass-limited samples. Capillary electrophoresis (CE) is a promising separation technique for top-down proteomics due to its high resolution, high sensitivity, and short cycle times compared to traditional liquid chromatography (LC)-based methods. We recently developed the "Spray-Capillary," an ESI-assisted device for quantitative ultralow-volume sampling and online CE-MS analysis, which successfully characterized hundreds of intact proteoforms from picogram-level cell lysate samples and showed promise for quantitative analysis of mass-limited complex biological samples. In this study, we further improved throughput for mass-limited top-down proteomics by integrating multisegment sample injection with our spray-capillary CE-MS analysis platform. By optimizing the spacer between sample plugs, we enabled the injection of multiple samples into the spray-capillary prior to a single CE-MS analysis, achieving baseline separation of identical proteins from different segments. Under optimized conditions, we quantifiedE. colilysate (10-250 pg) using a six-point calibration curve in a single analysis (e.g., ∼60 min run time), yielding a strong linear correlation (R2 > 0.98). Our method supports up to 17 sample segments per run (e.g., ∼90 min run time) while maintaining baseline separation. This optimized multisegment injection platform has the potential to analyze hundreds of mass-limited samples (e.g., single cells) per day, significantly enhancing throughput in top-down proteomics.

Precise characterization of proteins and proteoforms within the protein corona is essential for developing safer and more effective nanomedicines for diagnostic and therapeutic applications. Although the protein corona phenomenon has been recognized in nanomedicine for nearly two decades, the application of top-down proteomics to analyze proteoforms within this context has only recently gained traction. In this study, we advance proteoform-level analysis of the protein corona by integrating mass spectrometry (MS)-based top-down proteomics (TDP) and bottom-up proteomics (BUP). TDP analysis of protein corona of polystyrene nanoparticles (PSNPs) identified 3,505 proteoforms of 344 genes in human plasma samples, representing nearly 4-fold improvement in the number of proteoform and gene identifications (IDs) from protein corona of PSNPs and the largest proteoform dataset of protein corona reported so far. BUP analysis of the protein coronas identified 4,570 protein groups, 45,790 peptides, and 23,632 peptides containing modifications in the human plasma samples, representing one of the most comprehensive plasma proteome datasets from BUP to date and over 150% increase in protein IDs compared to previous PSNP–based corona studies. The combination of such large TDP and BUP datasets improves the characterization quality of nearly 35% of identified proteoforms containing mass shifts, producing a more precise proteoform landscape of protein corona. This BUP and TDP combination approach exceeds the capabilities of individual techniques for proteoform characterization in protein corona, and will eventually enhance our understanding of the protein corona and offer valuable insights into nanoparticle–biosystem interactions, as well as advancing proteoform-level biomarker discovery.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and pathological protein aggregation. Comprehensive quantitative proteomics of brain tissues from AD patients is critical for pursuing a better understanding of the molecular mechanisms that drive AD progression. Here, we present one of the first quantitative top-down proteomics (TDP) studies of postmortem cortex samples from AD patients and healthy controls to profile their proteoform differences by coupling capillary zone electrophoresis (CZE, liquid-phase ion mobility) to tandem mass spectrometry (MS/MS). We identified 3191 unique proteoforms and uncovered a drastic transformation in the proteoform profile in AD compared to healthy controls. Over 2200 proteoforms were exclusively identified in either AD or healthy control samples, and 157 proteoforms identified in both AD and control samples showed statistically significant abundance differences between the two conditions. Gene Ontology and pathway analysis of the genes associated with those proteoforms revealed broad changes in biological processes in AD brains, for example, telomere organization, substantia nigra development, amyloid fibril formation, microtubule cytoskeleton organization, progressive neurological disorders, long-term synaptic potentiation, and axogenesis. These biological processes are highly associated with the development of AD. Our study revealed a pool of potential novel proteoform biomarkers of AD in human brain samples for early diagnosis and therapy development. SUMMARY: Alzheimer's disease (AD) is a chronic neurodegenerative disease, destroying brain cells and causing thinking ability and memory to decline over time. Proteins (e.g., amyloid and tau) play key roles in the development of AD. Global and accurate protein measurement of human brains of AD patients and healthy controls will shed new light on the molecular mechanisms driving AD progression and discover new biomarkers for AD diagnosis and therapeutic development. Here, we performed the first CZE-MS/MS-based quantitative top-down proteomics (TDP) of a small cohort of AD human brain samples and healthy controls (5 AD and 5 control) to determine the differentially quantified proteoforms between the two health conditions. Over 3000 proteoforms were identified, and only about 700 proteoforms were detected in both conditions, indicating drastically different proteoform profiles between the two conditions. The differentially quantified proteoforms (e.g., tau, neurogranin, and calmodulin-1 proteoforms) are associated with biological processes relevant to AD development, for example, amyloid fibril formation, microtubule disruption, synaptic transmission, and axogenesis. The results offer a deep view of the proteoform transformation in the AD human brain compared to the healthy control, providing potential proteoform biomarkers for AD diagnosis and proteoform targets for therapeutic development.

A central challenge in top-down proteomics is the characterization of large proteoforms (>70 kDa) due to their high spectral complexity in mass spectrometers. Here, we advance individual ion mass spectrometry (I2MS) for intact mass and fragmentation analysis of α- and β-catenins (85-110 kDa), key components of adherens junctions. Using denatured I2MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on α-catenin and 7 on β-catenin, with site-specific changes corresponding to actomyosin contractility. Notably, phosphorylation at α-catenin S641 was constitutive, while other sites in the P-linker and actin-binding domains as well as β-catenin S675 and S552 were sensitive to actomyosin perturbation. Application of I2MS for fragment ion detection (I2MS2) also enabled 25-30% sequence coverage for these exceptionally large proteoforms, compared to <1% using conventional methods for top-down mass spectrometry. Our results support a catenin phospho-code model, wherein combinatorial phosphorylation patterns encode mechano-transductive signals regulating cell-cell adhesion. This work establishes top-down I2MS as a viable approach for probing complex post-translational modification landscapes in high-mass proteins and highlights proteoforms as functional units in cellular regulation.

Detection and Quantitation of Small Proteins Using Mass Spectrometry.

Top-down proteomics (TDP) is a powerful approach for characterizing intact protein molecules and their diverse proteoforms. Despite recent advances, current TDP software tools often suffer from fragmented workflows, steep learning curves for non-experts, or limited interactive visualization capabilities. To address these challenges, we introduce TDEase, an integrated analytical framework designed to streamline and enhance TDP data interpretation, with a current focus on integration with the TopPIC suite package for targeted proteoform characterization. TDEase features a modular architecture comprising TDPipe, a multi-process data processing engine, and TDVis, an interactive web-based visualization module. TDPipe automates the execution of mainstream TDP analysis algorithms through a user-configurable pipeline, ensuring seamless and reproducible data processing. The TDVis module then transforms these results into dynamic, interactive dashboards, enabling multidimensional data exploration, including feature maps and PTM analysis. An alternative version, TDVisWeb, is also available for visualizing the results on an internet server or intranet workstation at institutional core facilities. We demonstrated the software capabilities in proteoform identification and comparative analysis using published histone datasets. TDEase is built with Python and open-source, allowing future improvements and incorporation of more data types as the TDP community develops new software. Source code is available at https://github.com/Computational-TDMS/TDEase.

Bottom-up proteomics introduces proteoform ambiguitydue to theloss of connectivity between peptides and their original proteoforms.Top-down proteomics (TDP) removes the ambiguity through the directidentification and characterization of intact proteoforms and theirrespective post-translational modifications (PTM). Electron capturedissociation (ECD) is an efficient and gentle peptide and proteinfragmentation strategy that can be used for both bottom-up and top-downapproaches. Here, we used an Agilent 6550 Q-TOF mass spectrometerretrofitted with an e-MSion ECD cell. Top-down sequencing capabilitiesof the cell were evaluated by sequencing of intact peptides and proteinson high-performance liquid chromatography (HPLC) time scales. Amyloidbeta 1-40 (Aβ40) was first tested due to its pathophysiologicalrole in Alzheimer’s disease and served as our large peptidestandard. We sequenced Aβ40 via reverse-phase HPLC-MS and achieved95% sequence coverage on chromatographic time scales utilizing a data-dependentacquisition (DDA)-based method. Acetone-precipitated protein extractsfrom human brain were then separated by HPLC and analyzed with a DDAmethod, which identified 16 proteoforms between 2 and 17 kDa withsequence coverage ranging from 7 to 90% based on proteoform size andcomposition. In addition to proteoform identification, ECD fragmentationdistinguished multiple isoaspartate modifications from aspartate,as well as accurately differentiating leucine from isoleucine residuesdirectly from the human brain sample. Here, we observed isoaspartatewithin a thymosin beta-4 proteoform. Additionally, we demonstratedthe differentiation of leucine and isoleucine within a subunit ofubiquitin. This study advances the application of LC-Q-TOF instrumentationfor discovery-based top-down proteomics utilizing ECD as enabled bythe e-MSion ECD cell.