Enhance proteoform sequence coverage by using top-down proteomics and middle-proteomics
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Equus Caballus (horse) Bos Taurus (bovine)
SUBMITTER: 
Xingzhao Xiong
LAB HEAD: Xiaowen (Kevin) Liu
PROVIDER: PXD068831 | Pride | 2026-02-17
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 320 |
Similar Datasets
Project description:We applied a middle-down proteomics strategy for large scale protein analysis during in vivo development of Caenorhabditis elegans. We characterized post-translational modifications (PTMs) on histone H3 N-terminal tails at eight time points during the C. elegans lifecycle, including embryo, larval stages (L1 to L4), dauer and L1/L4 post dauer. Histones were analyzed by our optimized middle-down protein sequencing platform using high mass accuracy tandem mass spectrometry. This allows quantification of intact histone tails and detailed characterization of distinct histone tails carrying co-occurring PTMs. We measured temporally distinct combinatorial PTM profiles during C. elegans development. We show that the doubly modified form H3K23me3K27me3, which is rare or non-existent in mammals, is the most abundant PTM in all stages of C. elegans lifecycle. The abundance of H3K23me3 increased during development and it was mutually exclusive of the active marks H3K18ac, R26me1 and R40me1, suggesting a role for H3K23me3 in to silent chromatin. We observed distinct PTM profiles for normal L1 larvae and for L1-post dauer larvae, or L4 and L4 post-dauer, suggesting that histone PTMs mediate an epigenetic memory that is transmitted during dauer formation. Collectively, our data describe the dynamics of histone H3 combinatorial code during C. elegans lifecycle and demonstrate the feasibility of using middle-down proteomics to study in vivo development of multicellular organisms.
2015-11-02 | PXD002525 | Pride
Project description:Mass spectrometry (MS)-based top-down characterization of integral membrane proteins (IMPs) is crucial for understanding their functions in biological processes. However, it is technically challenging due to their low solubility in typical MS-compatible buffers. In this work, for the first time, we developed an efficient capillary zone electrophoresis (CZE)-tandem MS (MS/MS) method for top-down proteomics (TDP) of IMPs enriched from mouse brains. Our technique employs a sample buffer containing 30% (v/v) formic acid and 60% (v/v) methanol for solubilizing IMPs and utilizes a separation buffer of 30% (v/v) acetic acid and 30% (v/v) methanol for maintaining the solubility of IMPs during CZE separation. Single-shot CZE-MS/MS identified 51 IMP proteoforms from the mouse brain sample. Coupling size exclusion chromatography (SEC) to CZE-MS/MS enabled the identification of 276 IMP proteoforms from the mouse brain sample and those proteoforms contained 1-4 transmembrane domains. This proof-of-concept work demonstrates the high potential of CZE-MS/MS for large-scale TDP of IMPs.
2023-12-07 | PXD042298 | Pride
Project description:Senescent cells are increasingly recognized as major contributors to age-related diseases, primarily through the secretion of bioactive molecules known as the senescence-associated secretory phenotype (SASP). The SASP plays a crucial role in promoting various age-related conditions, including neurodegeneration and type-2 diabetes. While bottom-up proteomic profiling has been instrumental in elucidating senescence heterogeneity and linking SASP factors to aging and obesity, the complexity of protein modifications in SASP remains underexplored. Notably, proteoform-level analysis has not yet been applied to study SASP profiles in detail. In this study, we employ top-down proteomics to capture proteoform diversity in the secretomes of senescent and healthy lung fibroblasts (IMR90).
2025-12-29 | PXD061829 | Pride
Project description:Large-scale top-down proteomics characterizes proteoforms in cells globally with high confidence and high throughput using reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) or capillary zone electrophoresis (CZE)-MS/MS. The false discovery rate (FDR) from the target-decoy database search is typically deployed to filter identified proteoforms to ensure high-confidence identifications (IDs). It has been demonstrated that the FDRs in top-down proteomics can be drastically underestimated. An alternative approach to the FDR can be useful for further evaluating the confidence of proteoform IDs after database search. We argue that predicting retention/migration time of proteoforms from the RPLC/CZE separation accurately and comparing their predicted and experimental separation time could be a useful and practical approach. Based on our knowledge, there is still no report in the literature about predicting separation time of proteoforms using large top-down proteomics datasets. In this pilot study, for the first time, we evaluated various semi-empirical models for predicting proteoforms’ electrophoretic mobility (µef) using large-scale top-down proteomics datasets from CZE-MS/MS. We achieved a linear correlation between experimental and predicted µef of E. coli proteoforms (R2=0.98) with a simple semi-empirical model, which utilizes the number of charges and molecular mass of each proteoform as the parameters. Our modeling data suggest that the complete unfolding of proteoforms during CZE separation benefits the prediction of their µef. Our results also indicate that N-terminal acetylation and phosphorylation both decrease proteoforms’ charge by roughly one charge unit.
2020-02-17 | PXD017265 | Pride
Project description:Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E-values) of proteoforms than CZE-HCD and CZE-ETD, indicating higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n=3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum-level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.
2019-06-11 | PXD012247 | Pride
Project description:We present an orthogonal multidimensional separation platform that couples size exclusion chromatography (SEC) and RPLC based protein prefractionation to CZE-MS/MS for deep top-down proteomics of Escherichia coli. The platform generated high peak capacity (∼4000) for separation of intact proteins, leading to the identification of 5700 proteoforms from the Escherichia coli proteome.
2025-12-15 | PXD071282 | Pride
Project description:Mass spectrometry (MS)-based spatially resolved top-down proteomics (TDP) of tissues is crucial for understanding the roles played by microenvironmental heterogeneity in the biological functions of organs and for discovering new proteoform biomarkers of diseases. There are few published spatially resolved TDP studies. One of the challenges relates to the limited performance of TDP for the analysis of spatially isolated samples using, for example, laser capture microdissection (LCM) because those samples are usually mass-limited. We present the first pilot study of LCM-capillary zone electrophoresis (CZE)-MS/MS for spatially resolved TDP and used zebrafish brain as the sample. The LCM-CZE-MS/MS platform employed a non-ionic detergent and a freeze-thaw method for efficient proteoform extraction from LCM isolated brain sections followed by CZE-MS/MS without any sample cleanup step, ensuring high sensitivity. Over 400 proteoforms were identified in a CZE-MS/MS analysis of one LCM brain section via consuming the protein content of roughly 250 cells. We observed drastic differences in proteoform profiles between two LCM brain sections isolated from the optic tectum (Teo) and telencephalon (Tel) regions. Proteoforms of three proteins (npy, penkb, and pyya) having neuropeptide hormone activity were exclusively identified in the isolated Tel section. Proteoforms of reticulon, myosin, and troponin were almost exclusively identified in the isolated Teo section, and those proteins play essential roles in visual and motor activities. The proteoform profiles accurately reflected the main biological functions of the Teo and Tel regions of the brain. Additionally, hundreds of post-translationally modified proteoforms were identified.
2023-03-11 | PXD027741 | Pride
Project description:Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics from CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction based CZE-MS/MS system approached 1-µL loading capacity, 90-min separation window and high peak capacity (~280) for separation of Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. About 2,800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins were identified from an Escherichia coli lysate by single-shot CZE-MS/MS with spectrum-level false discovery rate (FDR) less than 1%. The number of proteoforms is over three times higher than that from previous single-shot CZE-MS/MS.
2017-10-30 | PXD007273 | Pride
Project description:Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by cognitive decline and pathological protein aggregation. Comprehensive protein characterization of human brains of AD patients and comparison with healthy samples is critical for pursuing a better understanding of the molecular mechanisms that drive AD progression. Although many large-scale bottom-up proteomics studies of AD brains have been conducted, there are limited top-down proteomics (TDP) studies of human AD brains. This study employs CZE-MS/MS-based label-free quantitative TDP to profile proteoform differences in postmortem brain tissues from AD patients and healthy controls. Using a robust size exclusion chromatography (SEC) fractionation-CZE-MS/MS platform, we identified 3,191 unique proteoforms, uncovering distinct proteoform signatures between AD and healthy subjects. Principal component analysis (PCA) and hierarchical clustering revealed clear segregation of AD samples, driven by disease-specific changes in proteoform abundance . Notably, AD tissues exhibited elevated phosphorylation and combinatorial post-translational modifications (PTMs), including hyperphosphorylated tau and modified neurogranin proteoforms, which are implicated in synaptic dysfunction and neurodegeneration. Bidirectional regulation of proteoforms from key proteins such as Calmodulin-1 and MAPT further highlights the functional divergence in AD pathogenesis. Pathway analysis linked upregulated proteoforms in AD to amyloid fibril formation and microtubule disruption, while healthy samples were enriched for synaptic transmission and axogenesis pathways. These findings underscore the critical role of proteoform-specific alterations in AD pathology, offering new insights into molecular mechanisms and potential therapeutic targets.
2025-11-21 | PXD065459 | Pride
Project description:A universal and standardized sample preparation method becomes vital for denaturing top-down proteomics (dTDP) to advance the scale and accuracy of proteoform delineation in complex biological systems. It needs to have high protein recovery, minimum bias, good reproducibility, and compatibility with downstream mass spectrometry (MS) analysis. Here we employed a lysis buffer containing sodium dodecyl sulfate (SDS) for extracting proteoforms from cells, and for the first time, compared membrane ultrafiltration (MU), chloroform-methanol precipitation (CMP), and single-spot solid-phase sample preparation using magnetic beads (SP3) for proteoform cleanup for dTDP. The MU method outperformed CMP and SP3 methods, resulting in high and reproducible protein recovery from both E. coli cell (59±3%) and human HepG2 cell (86±5%) samples without a significant bias. Single-shot capillary zone electrophoresis (CZE)-MS/MS analyses of the prepared E. coli and HepG2 cell samples using the MU method identified 821 and 516 proteoforms, respectively. Nearly 30% and 50% of the identified E. coli and HepG2 proteins are membrane proteins. CZE-MS/MS identified 94 histone proteoforms from the HepG2 sample with various post-translational modifications, including acetylation, methylation, and phosphorylation. Our results suggest that combining the SDS-based protein extraction and the MU-based protein cleanup could be a universal sample preparation method for dTDP.
2020-05-22 | PXD018248 | Pride