Browsing by Subject "Top-down proteomics"

Now showing 1 - 7 of 7
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    Capillary zone electrophoresis-tandem mass spectrometry with activated ion electron transfer dissociation for large-scale top-down proteomics
    (Springer, 2019-12) McCool, Elijah N.; Basharat, Abdul Rehman; Liu, Xiaowen; Coon, Joshua J.; Sun, Liangliang; BioHealth Informatics, School of Informatics and Computing
    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 a 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.
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    Computational Methods for Proteoform Identification and Characterization Using Top-Down Mass Spectrometry
    (2023-12) Chen, Wenrong; Yan, Jingwen; Wang, Juexin; Wan, Jun; Zang, Yong; Luo, Xiao; Liu, Xiaowen
    Proteoforms, distinct molecular forms of proteins, arise due to numerous factors such as genetic mutations, differential gene expression, alternative splicing, and a range of biological processes. These proteoforms are often characterized by primary structural variances such as amino acid substitutions, terminal truncations, and post-translational modifications (PTMs). Proteoforms from the same proteins can manifest varied functional behaviors based on the specific alterations. The complexity inherent to proteoforms has elevated the significance of top-down mass spectrometry (MS) due to its proficiency in providing intricate sequence information for these intact proteoforms. During a typical top-down MS experiment, intact proteoforms are separated through platforms like liquid chromatography (LC) or capillary zone electrophoresis (CZE) prior to tandem mass spectrometry (MS/MS) analysis. Despite advancements in instruments and protocols for top-down MS, computational challenges persist, with software tool development still in its early stage. In this dissertation, our research revolves around three primary goals, all aimed at refining proteoform characterization. First, we bridge RNA-Seq with top-down MS for a better proteoform identification. We propose TopPG, an innovative proteogenomic tool which is tailored to generate proteoform sequence databases from genetic and splicing variations explicitly for top-down MS in contrast to traditional approaches. Second, to boost the accuracy of proteoform detection, we utilize machine learning methods to predict proteoform retention and migration times in top-down MS, an area previously overshadowed by bottom-up MS paradigms. critically evaluating models in a realm traditionally dominated by bottom-up MS methodologies. Lastly, recognizing the indispensable role of post-translational modifications (PTMs) on cellular functions, we introduce PTM-TBA. This tool integrates the complementary strengths of both top-down and bottom-up MS, augmented with annotations, building a comprehensive strategy for precise PTM identification and localization.
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    Deep Intact Proteoform Characterization in Human Cell Lysate using High-pH and Low-pH Reversed-Phase Liquid Chromatography
    (American Chemical Society, 2019-12) Yu, Dahang; Wang, Zhe; Sutton, Kellye A.; Liu, Xiaowen; Wu, Si; Computer and Information Science, School of Science
    Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low abundance PTM modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics.
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    Identification and Quantification of Proteoforms by Mass Spectrometry
    (Wiley, 2019-05) Schaffer, Leah V.; Millikin, Robert J.; Miller, Rachel M.; Anderson, Lissa C.; Fellers, Ryan T.; Ge, Ying; Kelleher, Neil L.; LeDuc, Richard D.; Liu, Xiaowen; Payne, Samuel H.; Sun, Liangliang; Thomas, Paul M.; Tucholski, Trisha; Wang, Zhe; Wu, Si; Wu, Zhijie; Yu, Dahang; Shortreed, Michael R.; Smith, Lloyd M.; BioHealth Informatics, School of Informatics and Computing
    A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, we outline some recent advances and discuss current challenges and future directions for the field.
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    Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry
    (MyJove Corporation, 2018-10-24) McCool, Elijah N.; Lubeckyj, Rachele; Shen, Xiaojing; Kou, Qiang; Liu, Xiaowen; Sun, Liangliang; Computer and Information Science, School of Science
    Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.
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    Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample
    (American Chemical Society, 2024) Sadeghi, Seyed Amirhossein; Chen, Wenrong; Wang, Qianyi; Wang, Qianjie; Fang, Fei; Liu, Xiaowen; Sun, Liangliang; Biomedical Engineering and Informatics, Luddy School of Informatics, Computing, and Engineering
    Mass spectrometry (MS)-based top-down proteomics (TDP) has revolutionized biological research by measuring intact proteoforms in cells, tissues, and biofluids. Capillary zone electrophoresis-tandem MS (CZE-MS/MS) is a valuable technique for TDP, offering a high peak capacity and sensitivity for proteoform separation and detection. However, the long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which is a crucial aspect for large-scale studies. This work investigated the long-term qualitative and quantitative reproducibility of CZE-MS/MS for TDP for the first time, focusing on a yeast cell lysate. Over 1000 proteoforms were identified per run across 62 runs using one linear polyacrylamide (LPA)-coated separation capillary, highlighting the robustness of the CZE-MS/MS technique. However, substantial decreases in proteoform intensity and identification were observed after some initial runs due to proteoform adsorption onto the capillary inner wall. To address this issue, we developed an efficient capillary cleanup procedure using diluted ammonium hydroxide, achieving high qualitative and quantitative reproducibility for the yeast sample across at least 23 runs. The data underscore the capability of CZE-MS/MS for large-scale quantitative TDP of complex samples, signaling its readiness for deployment in broad biological applications. The MS RAW files were deposited in ProteomeXchange Consortium with the data set identifier of PXD046651.
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    Quantitative Top-Down Proteomics in Complex Samples Using Protein-Level Tandem Mass Tag Labeling
    (American Chemical Society, 2021-06-02) Yu, Dahang; Wang, Zhe; Cupp-Sutton, Kellye A.; Guo, Yanting; Kou, Qiang; Smith, Kenneth; Liu, Xiaowen; Wu, Si; BioHealth Informatics, School of Informatics and Computing
    Labeling approaches using isobaric chemical tags (e.g., isobaric tagging for relative and absolute quantification, iTRAQ and tandem mass tag, TMT) have been widely applied for the quantification of peptides and proteins in bottom-up MS. However, until recently, successful applications of these approaches to top-down proteomics have been limited because proteins tend to precipitate and “crash” out of solution during TMT labeling of complex samples making the quantification of such samples difficult. In this study, we report a top-down TMT MS platform for confidently identifying and quantifying low molecular weight intact proteoforms in complex biological samples. To reduce the sample complexity and remove large proteins from complex samples, we developed a filter-SEC technique that combines a molecular weight cutoff filtration step with high-performance size exclusion chromatography (SEC) separation. No protein precipitation was observed in filtered samples under the intact protein-level TMT labeling conditions. The proposed top-down TMT MS platform enables high-throughput analysis of intact proteoforms, allowing for the identification and quantification of hundreds of intact proteoforms from Escherichia coli cell lysates. To our knowledge, this represents the first high-throughput TMT labeling-based, quantitative, top-down MS analysis suitable for complex biological samples.