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Neil L. Kelleher - One of the best experts on this subject based on the ideXlab platform.

  • A comprehensive pipeline for translational Top-down Proteomics from a single blood draw
    Nature Protocols, 2019
    Co-Authors: Timothy K. Toby, Luca Fornelli, Kristina Srzentić, Caroline J. Dehart, Josh Levitsky, John Friedewald, Neil L. Kelleher
    Abstract:

    Top-down Proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up Proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography–tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h. In Top-down Proteomics, intact proteins are analyzed by mass spectrometry. Toby et al. describe an approach for analyzing peripheral blood mononuclear cells by Top-down Fourier-transform MS, with data analysis using ProSight Lite and TDPortal.

  • top down Proteomics reveals mature proteoforms expressed in subcellular fractions of the echinococcus granulosus preadult stage
    Journal of Proteome Research, 2015
    Co-Authors: Karina Rodrigues Lorenzatto, Paul M. Thomas, Neil L. Kelleher, Kyunggon Kim, Ioanna Ntai, Gabriela Prado Paludo, Jeferson Camargo De Lima, Henrique Bunselmeyer Ferreira
    Abstract:

    Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up Proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC–MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentiall...

  • The emergence of Top-down Proteomics in clinical research
    Genome Medicine, 2013
    Co-Authors: John P Savaryn, Paul M. Thomas, Adam D Catherman, Michael M Abecassis, Neil L. Kelleher
    Abstract:

    Proteomic technology has advanced steadily since the development of 'soft-ionization' techniques for mass-spectrometry-based molecular identification more than two decades ago. Now, the large-scale analysis of proteins (Proteomics) is a mainstay of biological research and clinical translation, with researchers seeking molecular diagnostics, as well as protein-based markers for personalized medicine. Proteomic strategies using the protease trypsin (known as bottom-up Proteomics) were the first to be developed and optimized and form the dominant approach at present. However, researchers are now beginning to understand the limitations of bottom-up techniques, namely the inability to characterize and quantify intact protein molecules from a complex mixture of digested peptides. To overcome these limitations, several laboratories are taking a whole-protein-based approach, in which intact protein molecules are the analytical targets for characterization and quantification. We discuss these Top-down techniques and how they have been applied to clinical research and are likely to be applied in the near future. Given the recent improvements in mass-spectrometry-based Proteomics and stronger cooperation between researchers, clinicians and statisticians, both peptide-based (bottom-up) strategies and whole-protein-based (Top-down) strategies are set to complement each other and help researchers and clinicians better understand and detect complex disease phenotypes.

  • Evaluation of the Compact High-Field Orbitrap for Top-down Proteomics of Human Cells
    Journal of proteome research, 2012
    Co-Authors: Dorothy R. Ahlf, Paul M. Thomas, Philip D. Compton, John C. Tran, Bryan P. Early, Neil L. Kelleher
    Abstract:

    Mass spectrometry based Proteomics generally seeks to identify and fully characterize protein species with high accuracy and throughput. Recent improvements in protein separation have greatly expanded the capacity of Top-down Proteomics (TDP) to identify a large number of intact proteins. To date, TDP has been most tightly associated with Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Here, we couple the improved separations to a Fourier-transform instrument based not on ICR but using the Orbitrap Elite mass analyzer. Application of this platform to H1299 human lung cancer cells resulted in the unambiguous identification of 690 unique proteins and over 2000 proteoforms identified from proteins with intact masses 500 identifications) in an Orbitrap mass spectrometer and exemplifies an accessible platform for whole protein mass spectrometry.

  • top down Proteomics on a chromatographic time scale using linear ion trap fourier transform hybrid mass spectrometers
    Analytical Chemistry, 2007
    Co-Authors: Bryan A Parks, Craig D Wenger, Michael T Boyne, Patricia V Burke, Kurt E Kwast, Michael J. Roth, Paul M. Thomas, Lihua Jiang, Neil L. Kelleher
    Abstract:

    Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, Top-down Proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography−tandem mass spectrometry (LC−MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC−MS/MS, we have detected 231 metabolically labeled (14N/15N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC−MS/MS of selected anion exchange fractions. Automa...

Xiaowen Liu - One of the best experts on this subject based on the ideXlab platform.

  • Quantitative Top-down Proteomics in Complex Samples Using Protein-Level Tandem Mass Tag Labeling.
    Journal of the American Society for Mass Spectrometry, 2021
    Co-Authors: Zhe Wang, Kellye A Cupp-sutton, Yanting Guo, Qiang Kou, Kenneth Smith, Xiaowen Liu
    Abstract:

    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.

  • mash explorer a universal software environment for top down Proteomics
    Journal of Proteome Research, 2020
    Co-Authors: David S Roberts, Jake A Melby, Xiaowen Liu, Kent Wenger, Molly Wetzel, Sudharshanan Govindaraj Ramanathan, Elizabeth F Bayne, Ruixiang Sun, Irene M Ong, Sean Mcilwain
    Abstract:

    Top-down mass spectrometry (MS)-based Proteomics enable a comprehensive analysis of proteoforms with molecular specificity to achieve a proteome-wide understanding of protein functions. However, the lack of a universal software for Top-down Proteomics is becoming increasingly recognized as a major barrier, especially for newcomers. Here, we have developed MASH Explorer, a universal, comprehensive, and user-friendly software environment for Top-down Proteomics. MASH Explorer integrates multiple spectral deconvolution and database search algorithms into a single, universal platform which can process Top-down Proteomics data from various vendor formats, for the first time. It addresses the urgent need in the rapidly growing Top-down Proteomics community and is freely available to all users worldwide. With the critical need and tremendous support from the community, we envision that this MASH Explorer software package will play an integral role in advancing Top-down Proteomics to realize its full potential for biomedical research.

  • Deep Intact Proteoform Characterization in Human Cell Lysate Using High-pH and Low-pH Reversed-Phase Liquid Chromatography
    Journal of The American Society for Mass Spectrometry, 2019
    Co-Authors: Zhe Wang, Kellye A Cupp-sutton, Xiaowen Liu
    Abstract:

    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.

  • deep top down Proteomics using capillary zone electrophoresis tandem mass spectrometry identification of 5700 proteoforms from the escherichia coli proteome
    PMC, 2018
    Co-Authors: Elijah N Mccool, Qiang Kou, Xiaowen Liu, Xiaojing Shen, Daoyang Chen, Rachele A Lubeckyj, Liangliang Sun
    Abstract:

    Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as a useful tool for Top-down Proteomics. However, its performance for deep Top-down Proteomics is still dramatically lower than widely used reversed-phase liquid chromatography (RPLC)-MS/MS. 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. The data represents a 10-fold improvement in the number of proteoform identifications compared with previous CZE-MS/MS studies and represents the largest bacterial Top-down Proteomics data set reported to date. The performance of the CZE-MS/MS based platform is comparable to the state-of-the-art RPLC-MS/MS based systems in terms of the number of prote...

  • single shot top down Proteomics with capillary zone electrophoresis electrospray ionization tandem mass spectrometry for identification of nearly 600 escherichia coli proteoforms
    Analytical Chemistry, 2017
    Co-Authors: Rachele A Lubeckyj, Qiang Kou, Xiaowen Liu, Xiaojing Shen, Elijah N Mccool, Liangliang Sun
    Abstract:

    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 using 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 the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction-based CZE-MS/MS approached a 1 μL loading capacity, 90 min separation window, and high peak capacity (∼280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for Top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified...

Liangliang Sun - One of the best experts on this subject based on the ideXlab platform.

  • Toward a Universal Sample Preparation Method for Denaturing Top-down Proteomics of Complex Proteomes.
    Journal of proteome research, 2020
    Co-Authors: Zhichang Yang, Xiaojing Shen, Daoyang Chen, Liangliang Sun
    Abstract:

    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 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 Escherichia 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. The MS raw data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD018248.

  • predicting electrophoretic mobility of proteoforms for large scale top down Proteomics
    Analytical Chemistry, 2020
    Co-Authors: Daoyang Chen, Zhichang Yang, Xiaojing Shen, Rachele A Lubeckyj, Elijah N Mccool, Qianjie Wang, Liangliang Sun
    Abstract:

    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/M...

  • deep top down Proteomics using capillary zone electrophoresis tandem mass spectrometry identification of 5700 proteoforms from the escherichia coli proteome
    PMC, 2018
    Co-Authors: Elijah N Mccool, Qiang Kou, Xiaowen Liu, Xiaojing Shen, Daoyang Chen, Rachele A Lubeckyj, Liangliang Sun
    Abstract:

    Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as a useful tool for Top-down Proteomics. However, its performance for deep Top-down Proteomics is still dramatically lower than widely used reversed-phase liquid chromatography (RPLC)-MS/MS. 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. The data represents a 10-fold improvement in the number of proteoform identifications compared with previous CZE-MS/MS studies and represents the largest bacterial Top-down Proteomics data set reported to date. The performance of the CZE-MS/MS based platform is comparable to the state-of-the-art RPLC-MS/MS based systems in terms of the number of prote...

  • single shot top down Proteomics with capillary zone electrophoresis electrospray ionization tandem mass spectrometry for identification of nearly 600 escherichia coli proteoforms
    Analytical Chemistry, 2017
    Co-Authors: Rachele A Lubeckyj, Qiang Kou, Xiaowen Liu, Xiaojing Shen, Elijah N Mccool, Liangliang Sun
    Abstract:

    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 using 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 the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction-based CZE-MS/MS approached a 1 μL loading capacity, 90 min separation window, and high peak capacity (∼280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for Top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified...

  • fast separation and analysis of reduced monoclonal antibodies with capillary zone electrophoresis coupled to mass spectrometry
    Talanta, 2016
    Co-Authors: Yimeng Zhao, Liangliang Sun, Michael D Knierman, Norman J Dovichi
    Abstract:

    Capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) was used for analysis of reduced antibodies. We first developed a simple protocol to condition commercial linear-polyacrylamide coated capillaries for use in Top-down Proteomics. We then suspended reduced antibodies in a solution of 35% acetic acid, 50% acetonitrile in water. Heavy and light chains were baseline resolved within 10 min and with 3-30 µg/mL detection limits using a 0.1% aqueous formic acid background electrolyte. Quintuplicate runs of a two-antibody mixture produced relative standard deviations of ∼1% in migration time and 10% in peak amplitudes. Resolution was further improved for the two-antibody mixture by using 5% acetic acid as the background electrolyte, highlighting the potential of capillary electrophoresis-mass spectrometry for analysis of antibody mixtures.

Joseph A. Loo - One of the best experts on this subject based on the ideXlab platform.

  • internal fragments generated by electron ionization dissociation enhance protein top down mass spectrometry
    Journal of the American Society for Mass Spectrometry, 2020
    Co-Authors: Muhammad A Zenaidee, Rachel Ogorzalek R Loo, Carter Lantz, Taylor Perkins, Wonhyuek Jung, Joseph A. Loo
    Abstract:

    Top-down Proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentat...

  • Top or Middle? Up or Down? Toward a Standard Lexicon for Protein Top-down and Allied Mass Spectrometry Approaches
    Journal of The American Society for Mass Spectrometry, 2019
    Co-Authors: Frederik Lermyte, Yury O. Tsybin, Peter B. O’connor, Joseph A. Loo
    Abstract:

    In recent years, there has been increasing interest in Top-down mass spectrometry (TDMS) approaches for protein analysis, driven both by technological advancements and efforts such as those by the multinational Consortium for Top-down Proteomics (CTDP). Today, diverse sample preparation and ionization methods are employed to facilitate TDMS analysis of denatured and native proteins and their complexes. The goals of these studies vary, ranging from protein and proteoform identification, to determination of the binding site of a (non)covalently-bound ligand, and in some cases even with the aim to study the higher order structure of proteins and complexes. Currently, however, no widely accepted terminology exists to precisely and unambiguously distinguish between the different types of TDMS experiments that can be performed. Instead, ad hoc developed terminology is often used, which potentially complicates communication of Top-down and allied methods and their results. In this communication, we consider the different types of Top-down (or Top-down-related) MS experiments that have been performed and reported, and define distinct categories based on the protocol used and type(s) of information that can be obtained. We also consider the different possible conventions for distinguishing between middle- and Top-down MS, based on both sample preparation and precursor ion mass. We believe that the proposed framework presented here will prove helpful for researchers to communicate about TDMS and will be an important step toward harmonizing and standardizing this growing field. Graphical Abstract

  • An integrated native mass spectrometry and Top-down Proteomics method that connects sequence to structure and function of macromolecular complexes
    Nature Chemistry, 2018
    Co-Authors: Hong Hanh Nguyen, Rachel R. Ogorzalek Loo, Iain D. G. Campuzano, Joseph A. Loo
    Abstract:

    Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while Proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and Top-down Proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate Proteomics and structural biology to provide insights into protein structure, function and regulation. An integrated native mass spectrometry and Top-down Proteomics method using Fourier transform ion cyclotron resonance has been developed for the characterization of macromolecular protein complexes. This approach directly yields primary to quaternary structural information in a single native Top-down experiment.

  • an integrated native mass spectrometry and top down Proteomics method that connects sequence to structure and function of macromolecular complexes
    Nature Chemistry, 2018
    Co-Authors: Hong Hanh Nguyen, Iain D. G. Campuzano, Rachel Ogorzalek R Loo, Joseph A. Loo
    Abstract:

    Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while Proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and Top-down Proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate Proteomics and structural biology to provide insights into protein structure, function and regulation.

Bifan Chen - One of the best experts on this subject based on the ideXlab platform.

  • Best practices and benchmarks for intact protein analysis for Top-down mass spectrometry
    Nature Methods, 2019
    Co-Authors: Daniel Donnelly, Luca Fornelli, Ziqing Lin, Catherine Rawlins, Caroline Dehart, Luis Schachner, Jennifer Lippens, Krishna Aluri, Richa Sarin, Bifan Chen
    Abstract:

    One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

  • a top down Proteomics platform coupling serial size exclusion chromatography and fourier transform ion cyclotron resonance mass spectrometry
    Analytical Chemistry, 2019
    Co-Authors: Trisha Tucholski, Bifan Chen, Samantha J Knott, Paige Pistono, Ziqing Lin
    Abstract:

    Mass spectrometry (MS) based Top-down Proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for Top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S:N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted Top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically ...

  • A Top-down Proteomics Platform Coupling Serial Size Exclusion Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
    2019
    Co-Authors: Trisha Tucholski, Bifan Chen, Samantha J Knott, Paige Pistono, Ziqing Lin
    Abstract:

    Mass spectrometry (MS) based Top-down Proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for Top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S:N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted Top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30–50 kDa) simultaneously in a single mass spectrum within a 100 m/z window. Notably, within a 5 m/z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30–50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted Top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures

  • top down Proteomics of large proteins up to 223 kda enabled by serial size exclusion chromatography strategy
    Analytical Chemistry, 2017
    Co-Authors: Wenxuan Cai, Bifan Chen, Andrew J Alpert, Trisha Tucholski, Sean Mcilwain, Takushi Kohmoto, Song Jin
    Abstract:

    Mass spectrometry (MS)-based Top-down Proteomics is a powerful method for the comprehensive analysis of proteoforms that arise from genetic variations and post-translational modifications (PTMs). However, Top-down MS analysis of high molecular weight (MW) proteins remains challenging mainly due to the exponential decay of signal-to-noise ratio with increasing MW. Size exclusion chromatography (SEC) is a favored method for size-based separation of biomacromolecules but typically suffers from low resolution. Herein, we developed a serial size exclusion chromatography (sSEC) strategy to enable high-resolution size-based fractionation of intact proteins (10–223 kDa) from complex protein mixtures. The sSEC fractions could be further separated by reverse phase chromatography (RPC) coupled online with high-resolution MS. We have shown that two-dimensional (2D) sSEC-RPC allowed for the identification of 4044 more unique proteoforms and a 15-fold increase in the detection of proteins above 60 kDa, compared to one-...

  • online hydrophobic interaction chromatography mass spectrometry for top down Proteomics
    Analytical Chemistry, 2016
    Co-Authors: Bifan Chen, Andrew J Alpert, Santosh G Valeja, Ying Peng, Ying Ge
    Abstract:

    Recent progress in Top-down Proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. ...