CoChat
Sign Up
Cited by 152Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsMetabolomics and Mass Spectrometry Studies

Top-down proteomics: challenges, innovations, and applications in basic and clinical research

Open access
Brown, Kyle A., Melby, Jake A., Roberts, David S., Ge, Ying·2020·Expert Review of Proteomics
DOI 10.1080/14789450.2020.1855982PMC PMC7864889
Open this paper in CoChat — free account

Abstract

Introduction- A better understanding of the underlying molecular mechanism of diseases is critical for developing more effective diagnostic tools and therapeutics toward precision medicine. However, many challenges remain to unravel the complex nature of diseases.Areas covered- Changes in protein isoform expression and post-translation modifications (PTMs) have gained recognition for their role in underlying disease mechanisms. Top-down mass spectrometry (MS)-based proteomics is increasingly recognized as an important method for the comprehensive characterization of proteoforms that arise from alternative splicing events and/or PTMs for basic and clinical research. Here, we review the challenges, technological innovations, and recent studies that utilize top-down proteomics to elucidate changes in the proteome with an emphasis on its use to study heart diseases.Expert opinion- Proteoform-resolved information can substantially contribute to the understanding of the molecular mechanisms underlying various diseases and for the identification of novel proteoform targets for better therapeutic development . Despite the challenges of sequencing intact proteins, top-down proteomics has enabled a wealth of information regarding protein isoform switching and changes in PTMs. Continuous developments in sample preparation, intact protein separation, and instrumentation for top-down MS have broadened its capabilities to characterize proteoforms from a range of samples on an increasingly global scale.

Integrity signals

Registry

Crossref · 10.1080/14789450.2020.1855982

Cites

163 of 163 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 188 cited sources checked · 0 supported · 0 flagged

  • Premisecitation

    Peptide-to-protein inference in bottom-up proteomics causes loss of proteoform information due to shared homologous regions and low sequence coverage.

    “However, the ‘peptide-to-protein inference’ problem in bottom-up proteomics leads to a loss in proteoform information because of many homologous sequence regions are shared between different protein forms and the generally low protein sequence coverage is observed [13,14].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] source

      Not checked in the free run.

    • cites [14] source

      Not checked in the free run.

  • Premisecitation

    Mutations, alternative splicing, and PTMs generate many proteoforms from a single gene, greatly diversifying the proteome.

    “Mutations, alternative splicing events, and post-translational modifications (PTMs) result in a host of proteoforms (a term referring to all the protein products that arise from a single gene) that greatly diversify the proteome [1–4].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [1] source

      Not checked in the free run.

    • cites [2] source

      Not checked in the free run.

    • cites [3] source

      Not checked in the free run.

    • cites [4] source

      Not checked in the free run.

  • Premisecitation

    Agar and colleagues reported best practices and benchmarks for top-down sample preparation in the Consortium of Top-down Proteomics.

    “To standardize sample preparation, a “best practices and benchmarks” was reported by Agar and colleagues in the Consortium of Top-down Proteomics [23].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] source

      Not checked in the free run.

  • Premisecitation

    MS-based proteomics is the most versatile and comprehensive strategy for protein characterization.

    “Mass spectrometry (MS)-based proteomics has emerged as the most versatile and comprehensive strategy for protein characterization [8,9].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [8] source

      Not checked in the free run.

    • cites [9] source

      Not checked in the free run.

  • Premisecitation

    Mutations and differential expression of protein isoforms drive molecular mechanisms underlying disease phenotypes.

    “Moreover, mutations and differential expression of protein isoforms have been shown to drive molecular mechanisms underlying disease phenotypes, necessitating their in-depth characterization [5,7].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] source

      Not checked in the free run.

    • cites [7] source

      Not checked in the free run.

  • Classified as probably not a substantial claim. 24
  • May combine two claims. 55
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In that approach, ultrafiltration spin filters exchange incompatible salts for low-concentration organic acid or volatile ammonium-based salts before analysis.

    “Using their outlined approach, incompatible salts and other components are removed from target proteins by ultrafiltration spin filters with specific molecular weight cutoffs to exchange MS-incompatible components for low concentration organic acid or volatile ammonium-based salts before analysis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Bottom-up proteomics is conventional because peptides are more soluble, easier to separate, and many bioinformatics tools exist.

    “This “bottom-up” proteomics approach is conventional as peptides, resulting from intact protein digestion, are biochemically more soluble, easier to separate, and there is numerous well-developed bioinformatics software available for protein identification and quantification [10].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Top-down proteomics analyzes intact proteins and is preferred for unambiguous proteoform-resolved detail.

    “For this reason, the “top-down” proteomics approach, which forgoes digestion to peptides and instead analyzes intact proteins, is the preferred method to achieve unambiguous, proteoform-resolved molecular detail (Figure 1) [15].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Brown et al. developed photocleavable ionic surfactant Azo, which has extraction efficiency comparable to SDS and is compatible with top-down MS after rapid UV degradation.

    “Brown et al. have recently developed a photocleavable ionic surfactant, 4-hexylphenylazosulfonate (Azo) [31], which shows comparable protein extraction efficiency to common surfactants such as SDS and was compatible with top-down MS analysis after rapid UV-degradation (Figure 2) [31].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Azo enables rapid tryptic digestion, acting as an all-in-one surfactant for integrated top-down and bottom-up proteomics.

    “Additionally, Azo was shown to enable rapid enzymatic digestion by trypsin, demonstrating its unique ability as an “all-in-one” surfactant for integrated top-down and bottom-up proteomics [36].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Precipitation may take time and introduce unwanted variability and loss of sample, since certain proteins do not dissolve again easily.

    “However, precipitation can be time-consuming and may add unintended sample variability and sample loss, as some proteins are not readily re-solubilized [26,27].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [26] source

      Not checked in the free run.

    • cites [27] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Native top-down MS uses non-denaturing conditions and often 100-200 mM ammonium acetate to analyze intact proteins and protein complexes.

    “Briefly, native top-down MS involves using non-denaturing conditions and often employing 100–200 mM ammonium acetate as a volatile buffer for the analysis of intact proteins and protein complexes.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [40] source

      Not checked in the free run.

    • cites [41] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In native MS of protein-micelle complexes, the complex is ionized and the protein is liberated from the surfactant micelle using high collisional energy.

    “In this case, the entire protein-micelle complex is ionized, and the protein is liberated from the surfactant micelle using high collisional energy in the mass spectrometer [49].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [49] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Native MS provides information on protein complex subunit stoichiometry, metal binding, protein interaction partners, and lipid binding.

    “Valuable information regarding protein complex subunit stoichiometry [42], metal-binding [43,44], protein interaction partners [45], and lipid binding [46] can be achieved using native MS.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [42] source

      Not checked in the free run.

    • cites [43] source

      Not checked in the free run.

    • cites [44] source

      Not checked in the free run.

    • cites [45] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    nanoPOTS is promising for highly sensitive top-down proteomics potentially including single-cell analysis, but most protein identifications were limited to sizes below 10 kDa.

    “The nanoPOTs technology, although still highly specialized, shows enormous promise to enable highly sensitive top-down proteomics (potentially even single-cell top-down analysis), but the majority of protein identifications were limited to sizes <10 kDa.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Monolithic columns offer high-resolution protein separation with low backpressure.

    “On the other hand, monolithic columns offer high-resolution protein separation with low backpressure [71].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [71] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Surface-silanized superparamagnetic magnetite particles coupled with GAPT-Zn improved synthetic scalability, reproducibility, and enrichment efficacy of these nanoparticles.

    “More recently, the synthetic scalability, reproducibility, and enrichment efficacy of these NPs were improved using surface-silanized superparamagnetic magnetite (Fe3O4) particles coupled with the GAPT-Zn ligand [58].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [58] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    cTnI is an important gold-standard cardiac biomarker.

    “cardiac troponin I (cTnI) [65–67], an important and gold-standard cardiac biomarker [68].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [68] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The NP-peptide approach enriched cTnI with high specificity (<1 ng/mL) and reproducibility while depleting highly abundant proteins such as human serum albumin.

    “The NP-peptide approach enabled the enrichment of cTnI with high specificity (< 1 ng/mL) and reproducibility, while at the same time depleted highly abundant proteins like human serum albumin.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [68] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Single-dimension RPLC can identify only ~100 intact proteoforms with limited coverage of larger species (>30 kDa).

    “Typically a single-dimension of RPLC can only enable the identification of ~100 intact proteoforms with limited coverage of larger species (>30 kDa) [69].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [69] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Antibodies have limitations including batch-to-batch variation, high production cost, poor chemical stability, and lack of proteoform-specific epitope recognition, which contribute to reproducibility issues.

    “However, antibodies possess many inherent limitations: batch-to-batch variation, high cost of production, poor chemical stability, and lack of proteoform-specific epitope recognition that collectively contribute to reproducibility issues [62–64].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [62] source

      Not checked in the free run.

    • cites [63] source

      Not checked in the free run.

    • cites [64] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Long columns >1 m provide peak capacity >400 and confidently identify ~900 proteoforms, but operate at ultrahigh pressures (~14,000 psi) and are not always practical.

    “Although long columns (>1 m) were demonstrated to provide a peak capacity of >400 and confidently identified ~900 proteoforms, they operate at ultrahigh pressures (~14,000 psi) and are not always practical [70].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [70] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Gargano et al. developed online capillary HILIC for top-down proteomics.

    “More recently, Gargano et al. developed online capillary HILIC for top-down proteomics [76].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [76] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    They used a C4 trap column for loading and doped propionic acid in the electrospray interface to overcome signal suppression of trifluoroacetic acid in the mobile phase.

    “Notably, they utilized a C4 trap column for loading (bypassing the traditional use of high organic solvent for sample composition) and doped propionic acid in the electrospray interface to overcome the signal suppression of trifluoroacetic acid in the mobile phase [76].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [76] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The Sun group optimized dynamic pH junction CZE using 10% acetic acid as background electrolyte to load ~1 μL and demonstrated single-shot CZE-MS identification of ~600 proteoforms from E. coli lysate.

    “To address these major issues, the Sun group optimized the dynamic pH junction CZE method, using 10% acetic acid as the background electrolyte, to load ~1 μL of sample and they demonstrated the use of single-shot CZE-MS analysis to identify ~600 proteoforms from an Escherichia coli lysate [74].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [74] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Chen et al. demonstrated online HIC-MS for determination of relative hydrophobicity, intact masses, and glycosylations of monoclonal antibodies and for gaining sequence and structural information using MS/MS.

    “Chen et al. demonstrated online HIC-MS analysis for determination of the relative hydrophobicity, intact masses, and glycosylations of monoclonal antibodies (mAb) as well as gaining sequence and structural information using MS/MS [81].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [81] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Online SEC has been used for rapid online desalting of antibodies and protein complexes.

    “For example, online size exclusion chromatography (SEC) has been used for the rapid online desalting of antibodies and protein complexes [77].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [77] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IEX-MS has been used to characterize protein conjugates.

    “Similarly, ion-exchange chromatography (IEX)-MS has been used to characterize protein conjugates [78,79].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [78] source

      Not checked in the free run.

    • cites [79] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    HILIC proteome coverage was limited and significant work remains before its full potential for top-down proteomics is recognized.

    “However, the proteome coverage was limited and significant work will still be needed before the full potential of HILIC for top-down proteomics is recognized.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [76] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Native SEC and CZE coupled identified 672 proteoforms, 144 proteins, and 23 protein complexes.

    “For example, native SEC and CZE were coupled to identify 672 proteoforms, 144 proteins, and 23 protein complexes [88].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [88] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Large proteins have inherently lower signal-to-noise and are underrepresented in top-down studies.

    “Often large proteins, which have an inherently lower signal-to-noise, are underrepresented in top-down studies [83].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [83] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PEPPI-MS was introduced as an alternative gel-based separation technique.

    “More recently, PEPPI (passively eluting protein from polyacrylamide gel as intact species)-MS was introduced as an alternative gel-based separation technique [85].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [85] source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    sSEC was developed by coupling columns with differing pore sizes and using an MS-compatible mobile phase (1% formic acid).

    “To bypass the use of SDS for separation, serial SEC (sSEC) was developed by coupling columns with differing pore sizes and using an MS-compatible mobile phase (1% formic acid).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Using PEPPI, good protein recovery and high-resolution separation were achieved for top-down proteomics of proteins <50 kDa.

    “Using PEPPI, good protein recovery, and high-resolution separation was achieved for top-down proteomics of protein <50 kDa.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [85] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    sSEC achieved high-resolution size-based separation of cardiac sarcomere proteins and, when used for offline fractionation before RPLC-MS, enabled detection of ~4,000 unique proteoforms up to 223 kDa.

    “sSEC was used to achieve high-resolution size-based separation of cardiac sarcomere proteins and, when used as for offline fractionation before RPLC-MS, enabled the detection of ~4,000 unique proteoforms up to 223 kDa [69].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [69] source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Offline protein fractionation remains labor-intensive, low-throughput, and requires relatively large sample quantities, making it unsuitable for sample-limited applications.

    “Despite the advancements in multidimensional separation strategies, performing offline protein fractionation remains labor-intensive, low-throughput, and requires relatively large sample quantities making it not suitable for sample limited applications.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    LESA directly samples proteins from a solid surface for analysis of proteins and protein complexes.

    “Another emerging technique is liquid extraction surface analysis (LESA), which directly sampling proteins from a solid surface for analysis of protein and protein complexes [94,95].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [94] source

      Not checked in the free run.

    • cites [95] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    q-TOF mass spectrometers are a common alternative, robust, affordable, and capable of analyzing relatively large protein species.

    “Quadrupole time-of-flight (q-TOF) mass spectrometers are a common alternative instrument platform, as they are robust, affordable, and capable of analyzing relatively large protein species [69,97].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [69] source

      Not checked in the free run.

    • cites [97] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ESI is predominantly utilized for top-down proteomics because it is easy to interface with common LC systems.

    “Although several ionization techniques are available, ESI is still predominately utilized for top-down proteomics as it is easy to interface with common LC systems [92].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [92] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Benchtop Orbitrap mass spectrometers are the most common instrument for proteomics, bypass cryogenic cooling, and offer high resolution and faster acquisition.

    “Currently, benchtop Orbitrap mass spectrometers are the most common instrument for proteomics as they bypass the need for cryogenic cooling (which is needed for the magnets of FTICR instruments) while still offering high-resolution and faster acquisition speeds [98,99].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [98] source

      Not checked in the free run.

    • cites [99] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    FTICR mass spectrometers are traditionally the instrument of choice for top-down because they resolve very large species, but slower scan speed can limit LC-MS/MS and global protein characterization.

    “Traditionally, Fourier-transform ion cyclotron resonance (FTICR) mass spectrometers are the instrument of choice for top-down because of its capabilities to resolve very large species [96]; however, the slower scan speed can limit its capabilities for effective LC-MS/MS analysis and global protein characterization.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [96] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Kafader et al. demonstrated increased sensitivity and sequence coverage when employing CDMS of intact protein analysis.

    “Additionally, Kafader et. al demonstrated increase sensitivity and sequence coverage when employing CDMS of intact protein analysis [107].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [107] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CDMS advantage is analysis of very large biomolecules including virus particles (2.47 MDa) and highly complex intact protein mixtures.

    “The major advantage of CDMS is the analysis of very large biomolecules including virus particles (2.47 MDa) and highly complex intact protein mixtures [108,109].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [108] source

      Not checked in the free run.

    • cites [109] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    PTR MS reduces the average number of charge states for improved signal-to-noise and reduced spectral complexity for identification of protein complex mixtures.

    “Similarly, proton transfer reaction (PTR) MS has been used to reduce the average number of charge states for improved signal-to-noise and reduced spectral complexity for the identification of protein complex mixtures [106].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [106] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    DDA in top-down LC-MS selects the topmost intense peaks in the initial MS scan for fragmentation.

    “Generally, data acquisition in top-down on an LC-MS timescale is performed using data-dependent acquisition (DDA) wherein the topmost intense peaks in the initial MS scan are selected for fragmentation [113].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [113] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    ECD and ETD breakages along the protein backbone occur more randomly, enabling higher sequence coverage while preserving labile PTMs.

    “Breakages along the protein backbone occur more randomly enabling higher sequence coverage while preserving labile PTMs.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [112] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In a cited study, AI-ETD identified 935 proteoforms, while HCD, ETD, and EThcD identified 1014, 915, and 817 proteoforms, respectively.

    “For example, Riley et al. demonstrated AI-ETD could identify 935 proteoforms while HCD, ETD, and EThcD identified 1014 proteoforms, 915, and 817, respectively [121].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [121] source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    UVPD predominantly breaks the bond between the alpha carbon and the carbonyl, yielding a/x ions, but can also produce b/y and c/z ions.

    “Predominately, UVPD breaks the bond between the alpha carbon and the carbonyl, resulting in a/x ions, but also can produce b/y and c/z ions.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Despite challenges of sequencing intact proteins, top-down proteomics has enabled a wealth of information regarding protein isoform switching and changes in PTMs.

    “Despite the challenges of sequencing intact proteins, top-down proteomics has enabled a wealth of information regarding protein isoform switching and changes in PTMs.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    UVPD's diversity of bond breakages makes it ideal for deep sequence coverage, but higher numbers of protein identifications are still obtained using HCD for global top-down analysis by LC-MS.

    “The diversity of bond breakages afforded by UVPD makes it ideal for deep sequence coverage, but higher numbers of protein identifications are still obtained using HCD for global top-down analysis by LC-MS [117].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [117] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CAD and HCD may cleave labile PTMs and cause unwanted neutral loss of water and ammonium.

    “However, because CAD and HCD often break the most labile bonds, it may cleave labile PTMs (e.g., phosphorylation) and cause unwanted neutral loss of water and ammonium [112].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [112] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Continuous developments in sample preparation, intact protein separation, and instrumentation for top-down MS have broadened its capabilities to characterize proteoforms from a range of samples on an increasingly global scale.

    “Continuous developments in sample preparation, intact protein separation, and instrumentation for top-down MS have broadened its capabilities to characterize proteoforms from a range of samples on an increasingly global scale.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based). May combine two claims.

    Interpretationnone given

    Proteoform-resolved information can substantially contribute to understanding molecular mechanisms of various diseases and identifying novel proteoform targets for therapeutic development toward precision medicine.

    “Proteoform-resolved information can substantially contribute to the understanding of the molecular mechanisms underlying various diseases and for the identification of novel proteoform targets for better therapeutic development toward precision medicine.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Many diseases have complex molecular mechanisms that remain poorly understood.
    • Rests on an assumption: Improved understanding of disease mechanisms is necessary for developing more effective diagnostics and therapeutics for precision medicine.

  • May combine two claims.

    Premisecitation

    EID uses high energy electrons for fragmentation and shows promise for improved internal fragmentation for higher sequence coverage.

    “Recently, electron ionization dissociation (EID), which uses high energy electrons for fragmentation, has shown promise for improved internal fragmentation for higher sequence coverage [116].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [116] Zenaidee M, 2020, Internal fragments generated by electron ionization dissociation enhances protein top-down mass spectrometry. J. Am. Soc. Mass Spectrom

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    UVPD preserves labile modifications and, unlike ECD and ETD, is charge state independent.

    “Ultraviolet photodissociation (UVPD) also preserves labile modification, but unlike ECD and ETD, it is charge state independent [20].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Slower reaction kinetics, protein-to-protein variability, and charge state dependence limit ECD and ETD for global proteomic analysis.

    “However, the slower reaction kinetics, protein-to-protein variability, and charge state dependence of these techniques limit their application for global proteomic analysis[112].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [112] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    UniDec was developed to handle highly heterogeneous spectra, which has been critical for interpreting native protein mass spectra.

    “UniDec was developed to handle highly heterogeneous spectra, which has been critical for the interpretation of native protein mass spectra [127].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [127] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    THRASH was the first software to automate deconvolution of high-resolution mass spectra and remains widely used.

    “Thorough High-Resolution Analysis of Spectra by Horn (THRASH) was the first software to automate the deconvolution of high-resolution mass spectra and is still widely used [124].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [124] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The increasing availability of different fragmentation tools has enabled high protein sequence coverage, improved global proteoform characterization, and characterization of higher-order protein structure.

    “Overall, the growing number of different fragmentation tools available has enabled high protein sequence coverage, improved global proteoform characterization, and characterization of higher-order protein structure [112].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [112] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    THRASH compares experimental envelopes to a theoretical isotopomer envelope based on average amino acid composition (averagine) and assigns a statistical score from the overlap.

    “THRASH uses a theoretical isotopomer envelope based on the average elemental composition of amino acids (averagine) [125] to compare to the experimental envelopes. A statistical score is then assigned based on the overlap of the experimental and theoretical fit.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [125] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    MS-Deconv generates many candidate isotopomer envelopes and scores sets of envelopes rather than a single fit.

    “On the other hand, MS-Deconv generates a large set of candidate isotopomer envelopes and scores sets of envelopes rather than a single fit [126].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [126] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MS/MS analysis has been used to study protein complexes and protein-ligand binding.

    “Beyond primary sequence coverage, MS/MS analysis has been utilized to study protein complexes [122] and protein-ligand binding [123].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [122] source

      Not checked in the free run.

    • cites [123] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    FLASHDeconv used pattern matching to deconvolute MS quickly, with speeds orders of magnitude higher than conventional approaches.

    “Recently, FLASHDeconv used pattern matching to quickly deconvolute MS with speeds orders of magnitudes higher than conventional approaches [128].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [128] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MS4 analysis can identify both the proteoform state and the associated ligand structure.

    “For example, Gault et al. demonstrated the use of MS4 analysis to identify both the proteoform state and the associated ligand structure [123].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [123] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Hypertrophic cardiomyopathy is a commonly heritable cardiovascular disease that thickens the ventricular septum and leads to obstructive heart failure if untreated.

    “Hypertrophic cardiomyopathy is a commonly heritable cardiovascular disease that results in the thickening of the ventricular septum, which results in obstructive heart failure if left untreated [147].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [147] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MASH Explorer integrates multiple spectral deconvolution and database searching algorithms into a single freely available universal platform that can process top-down proteomics data from various vendor formats.

    “recently the Ge group developed MASH Explorer, which integrates multiple spectral deconvolution and database searching algorithms into a single, freely-available universal platform that can process top-down proteomics data from various vendor formats [139].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [139] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MS-based proteomics has become a staple in biomedical research for discovering novel biomarkers and elucidating disease mechanisms.

    “MS-based proteomics has become a staple in biomedical research for the discovery of novel biomarkers and elucidation of disease mechanisms [140].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [140] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Around 1,400 mutations have been identified in 11 genes related to the sarcomere.

    “While around 1,400 mutations have been identified in 11 genes related to the sarcomere, the molecular events leading to disease phenotype remain largely misunderstood [148].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [148] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In the heart, protein isoform switching and PTMs regulate cardiac contraction and relaxation and contribute to heart failure progression.

    “For example, in the heart, protein isoform switching [141], and PTMs are known to regulate cardiac contraction and relaxation, as well as play a role in the progression of heart failure [61,142,143].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [141] source

      Not checked in the free run.

    • cites [61] source

      Not checked in the free run.

    • cites [142] source

      Not checked in the free run.

    • cites [143] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Circulating cTnI released into the blood after myocardial infarction is a known cardiac protein biomarker.

    “circulating cTnI released into the blood upon myocardial infarction is a known cardiac protein biomarker [144–146].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [144] source

      Not checked in the free run.

    • cites [145] source

      Not checked in the free run.

    • cites [146] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The correlation suggests protein crosstalk or dysregulation of signaling by cAMP-dependent protein kinase A.

    “Interestingly, the authors determined a strong linear correlation of decreased phosphorylation levels of cardiac troponin I and enigma homolog 2 in the HCM samples suggesting protein crosstalk or dysregulation of signaling by cAMP-dependent protein kinase A (Figure 4) [149].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [149] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    hiPSC-CMs offer clinical opportunities to study heritable cardiovascular diseases, test drug candidates, and enable cardiac regeneration and precision medicine.

    “While patient tissue samples are the most relevant for disease studies, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer unprecedented clinical opportunities to study heritable cardiovascular diseases, test various drug candidates, and enable cardiac regeneration and precision medicine [150].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [150] source

      Not checked in the free run.

    • Rests on an assumption: Patient tissue samples are the most relevant for disease studies.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Sarcopenia has detrimental effects on skeletal muscle due to deterioration of muscle mass.

    “The effects of aging, or sarcopenia, has detrimental effects on skeletal muscle due to the deterioration of muscle mass [152].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [152] source

      Not checked in the free run.

  • The quote only partly states this claim.

    Interpretationcitation

    Skeletal muscle is highly heterogeneous in its myofilament proteoforms.

    “Quantitative top-down proteomics discovered that there were distinct differences in the myofilament proteoforms found across the seven various rat skeletal muscle tissues, which demonstrates the highly heterogeneous nature of skeletal muscle (Figure 6) [154].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [154] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A range of clinical samples including serum, urine, or biopsy tissue have been studied using top-down proteomics to identify protein profiles characteristic of a disease and generate more reliable biomarkers.

    “A range of clinical samples including serum, urine, or biopsy tissue, have been studied using top-down proteomics to identify protein profiles characteristic of a certain disease generating a more reliable biomarker [155–157].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [155] source

      Not checked in the free run.

    • cites [156] source

      Not checked in the free run.

    • cites [157] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Analysis of antibodies by top-down can quickly determine modification state, drug conjugate ratio, quality control screening, and more.

    “For example, analysis of antibodies by top-down can quickly determine the modification state [81], drug conjugate ratio [161], quality control screening [162], and more [163,164].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [81] source

      Not checked in the free run.

    • cites [161] source

      Not checked in the free run.

    • cites [162] source

      Not checked in the free run.

71 of 71

  • The paper relies on this without support.

    Many diseases have complex molecular mechanisms that remain poorly understood.

    “However, many challenges remain to unravel the complex nature of diseases.”

  • The paper relies on this without support.

    Improved understanding of disease mechanisms is necessary for developing more effective diagnostics and therapeutics for precision medicine.

    “A better understanding of the underlying molecular mechanism of diseases is critical for developing more effective diagnostic tools and therapeutics towards precision medicine.”

  • The paper relies on this without support.

    hiPSC-CMs resemble fetal cardiomyocytes rather than fully matured cardiomyocytes.

    “However, a major limitation to hiPSC-CM resembles fetal CMs rather than fully matured CMs.”

  • The paper relies on this without support.

    There is a lack of comprehensive methods to measure the maturity of hiPSC-CMs.

    “In addition, there is a lack of comprehensive methods to measure the maturity of hiPSC-CMs.”

  • The paper relies on this without support.

    There remains an incomplete understanding of the molecular mechanism underlying sarcopenia.

    “However, there remains an incomplete understanding of the molecular mechanism underlying sarcopenia.”

  • The paper relies on this without support.

    Patient tissue samples are the most relevant for disease studies.

    “While patient tissue samples are the most relevant for disease studies, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer unprecedented clinical opportunities to study heritable cardiovascular diseases, test various drug candidates, and enable cardiac regeneration and precision medicine [150].”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Study date conflicts with its reference

    Minor

    The text dates the cited study to 2012, but reference 84 lists its publication year as 2011.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 5

  1. 10.1002/pro.5560031109 · DOI · Paper page
  2. 10.1021/cr068286z · DOI · Paper page
  3. Anderson NL, 2002, History Charact Diagn Prospects, 1, 845
  4. 10.1021/pr200258m · DOI · Paper page
  5. 10.1080/14789450.2016.1209414 · DOI · Paper page

Cited by

Sorted by citations

Related

Authors
Brown, K. A., Melby, J. A., Roberts, D. S., & Ge, Y.
Affiliations
University of Wisconsin–Madison (US)
Corresponding author
  • Brown, Kyle A.
  • Ge, Ying
ORCID
Venue
Expert Review of Proteomics
Publisher
Informa UK Limited
Published
2020-10-02
Volume
17
Issue
10
Pages
719-733
ISSN
  • 1478-9450 (ISSN-L)
  • 1744-8387
DOI
10.1080/14789450.2020.1855982
PMC
PMC7864889
OpenAlex
W3106602244
Cited by
152
References
163
Citations by year
2026: 23 · 2025: 37 · 2024: 28 · 2023: 26 · 2022: 19 · 2021: 18 · 2020: 1
FWCI
6.11
Type
journal article
Language
en
Open access
Locations
  • Expert Review of Proteomics (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
Foundation for the National Institutes of Health
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
Keywords
top-down proteomics, proteoforms, protein isoforms, post-translational modification, alternative splicing, heart disease, precision medicine, sample preparation, intact protein separation
MeSH terms
Heart Diseases, Humans, Protein Processing, Post-Translational, Proteomics, Tandem Mass Spectrometry

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-05.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review