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

In the paper

“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].”

What the sources say

• Partly supported

  The abstract (p. 1) reports peak capacities above 400 at 14,000 psi, and the Discussion (p. 11) reports assignment of approximately 900 proteoforms at 1% FDR. However, the paper does not link that assignment specifically to columns longer than 1 m. Its Methods (p. 2) say the 14,000-psi approach can be readily implemented on commercial systems; the Conclusions (p. 11) reserve the practical-instrument limitation for long columns packed with smaller particles that require still higher pressures. (LLM, conf=0.86)

  cites [70] sourceExperimental — accuracy not yet measured

In the paper

“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].”

What the sources say

• Partly supported

  The Introduction describes LESA extracting proteins directly from sampled surfaces, and the Conclusions report analysis of intact proteins from bacterial colonies. Our copy does not report LESA analysis of protein complexes; its mentions of complexes are in references to other studies. (LLM, conf=0.87)

  cites [94] sourceExperimental — accuracy not yet measured
• Supported

  The Introduction describes LESA sampling discrete locations on a surface with a solvent droplet before mass-spectrometry analysis of intact proteins. The Conclusions report successful LESA analysis of protein species on glass substrates, including protein complexes up to approximately 800 kDa. (LLM, conf=0.94)

  cites [95] source

In the paper

“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.”

What the sources say

• Supported

  The citation is attached to “averagine.” Results and Discussion (pp. 2–3) defines averagine as an average amino-acid composition, uses it to construct a theoretical isotopic profile, and describes statistical comparison with an experimental profile. (LLM, conf=0.90)

  cites [125] source

In the paper

“circulating cTnI released into the blood upon myocardial infarction is a known cardiac protein biomarker [144–146].”

What the sources say

• Not checked yetcites [144] source
• Supported

  The Introduction identifies cTnI as a cardiac-specific protein released into circulation upon cardiomyocyte injury and used as a diagnostic biomarker of myocardial infarction. The “High sensitivity cTnI platforms” section explicitly states that cTnI is released into circulation during MI. (LLM, conf=0.95)

  cites [145] source
• Not checked yetcites [146] source

In the paper

“The effects of aging, or sarcopenia, has detrimental effects on skeletal muscle due to the deterioration of muscle mass [152].”

What the sources say

• Supported

  The Sarcopenia section describes sarcopenia as an age-related decline in muscle mass, strength, and quality. The section on age-related changes in muscle contractility also links loss of muscle mass to loss of muscle force, supporting the citing sentence’s broad point. (LLM, conf=0.91)

  cites [152] source

In the paper

“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.”

What the sources say

• Not checked yetcites [23] source

In the paper

“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].”

What the sources say

• Not checked yetcites [10] source

In the paper

“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].”

What the sources say

• Not checked yetcites [15] source

In the paper

“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].”

What the sources say

• Not checked yetcites [31] source

In the paper

“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].”

What the sources say

• Not checked yetcites [36] source

In the paper

“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].”

What the sources say

• Not checked yetcites [26] source
• Not checked yetcites [27] source

In the paper

“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.”

What the sources say

• Not checked yetcites [40] source
• Not checked yetcites [41] source

In the paper

“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].”

What the sources say

• Not checked yetcites [49] source

In the paper

“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.”

What the sources say

• Not checked yetcites [42] source
• Not checked yetcites [43] source
• Not checked yetcites [44] source
• Not checked yetcites [45] source

In the paper

“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.”

What the sources say

• Not checked yetcites [38] source

In the paper

“On the other hand, monolithic columns offer high-resolution protein separation with low backpressure [71].”

What the sources say

• Not checked yetcites [71] source

In the paper

“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].”

What the sources say

• Not checked yetcites [58] source

In the paper

“cardiac troponin I (cTnI) [65–67], an important and gold-standard cardiac biomarker [68].”

What the sources say

• Not checked yetcites [68] source

In the paper

“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.”

What the sources say

• Not checked yetcites [68] source

In the paper

“Typically a single-dimension of RPLC can only enable the identification of ~100 intact proteoforms with limited coverage of larger species (>30 kDa) [69].”

What the sources say

• Not checked yetcites [69] source

In the paper

“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].”

What the sources say

• Not checked yetcites [62] source
• Not checked yetcites [63] source
• Not checked yetcites [64] source

In the paper

“More recently, Gargano et al. developed online capillary HILIC for top-down proteomics [76].”

What the sources say

• Not checked yetcites [76] source

In the paper

“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].”

What the sources say

• Not checked yetcites [76] source

In the paper

“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].”

What the sources say

• Not checked yetcites [74] source

In the paper

“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].”

What the sources say

• Not checked yetcites [81] source

In the paper

“For example, online size exclusion chromatography (SEC) has been used for the rapid online desalting of antibodies and protein complexes [77].”

What the sources say

• Not checked yetcites [77] source

In the paper

“Similarly, ion-exchange chromatography (IEX)-MS has been used to characterize protein conjugates [78,79].”

What the sources say

• Not checked yetcites [78] source
• Not checked yetcites [79] source

In the paper

“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.”

What the sources say

• Not checked yetcites [76] source

In the paper

“For example, native SEC and CZE were coupled to identify 672 proteoforms, 144 proteins, and 23 protein complexes [88].”

What the sources say

• Not checked yetcites [88] source

In the paper

“Often large proteins, which have an inherently lower signal-to-noise, are underrepresented in top-down studies [83].”

What the sources say

• Not checked yetcites [83] source

In the paper

“More recently, PEPPI (passively eluting protein from polyacrylamide gel as intact species)-MS was introduced as an alternative gel-based separation technique [85].”

What the sources say

• Not checked yetcites [85] source

In the paper

“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).”

In the paper

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

What the sources say

• Not checked yetcites [85] source

In the paper

“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].”

What the sources say

• Not checked yetcites [69] source

In the paper

“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.”

In the paper

“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].”

What the sources say

• Not checked yetcites [69] source
• Not checked yetcites [97] source

In the paper

“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].”

What the sources say

• Not checked yetcites [92] source

In the paper

“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].”

What the sources say

• Not checked yetcites [98] source
• Not checked yetcites [99] source

In the paper

“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.”

What the sources say

• Not checked yetcites [96] source

In the paper

“Additionally, Kafader et. al demonstrated increase sensitivity and sequence coverage when employing CDMS of intact protein analysis [107].”

What the sources say

• Not checked yetcites [107] source

In the paper

“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].”

What the sources say

• Not checked yetcites [108] source
• Not checked yetcites [109] source

In the paper

“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].”

What the sources say

• Not checked yetcites [106] source

In the paper

“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].”

What the sources say

• Not checked yetcites [113] source

In the paper

“Breakages along the protein backbone occur more randomly enabling higher sequence coverage while preserving labile PTMs.”

What the sources say

• Not checked yetcites [112] source

In the paper

“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].”

What the sources say

• Not checked yetcites [121] source

In the paper

“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.”

In the paper

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

In the paper

“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].”

What the sources say

• Not checked yetcites [117] source

In the paper

“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].”

What the sources say

• Not checked yetcites [112] source

In the paper

“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.”

In the paper

“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.”

In the paper

“Recently, electron ionization dissociation (EID), which uses high energy electrons for fragmentation, has shown promise for improved internal fragmentation for higher sequence coverage [116].”

What the sources say

• Not checked yetcites [116] Zenaidee M, 2020, Internal fragments generated by electron ionization dissociation enhances protein top-down mass spectrometry. J. Am. Soc. Mass Spectrom

In the paper

“Ultraviolet photodissociation (UVPD) also preserves labile modification, but unlike ECD and ETD, it is charge state independent [20].”

What the sources say

• Not checked yetcites [20] source

In the paper

“However, the slower reaction kinetics, protein-to-protein variability, and charge state dependence of these techniques limit their application for global proteomic analysis[112].”

What the sources say

• Not checked yetcites [112] source

In the paper

“UniDec was developed to handle highly heterogeneous spectra, which has been critical for the interpretation of native protein mass spectra [127].”

What the sources say

• Not checked yetcites [127] source

In the paper

“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].”

What the sources say

• Not checked yetcites [124] source

In the paper

“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].”

What the sources say

• Not checked yetcites [112] source

In the paper

“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].”

What the sources say

• Not checked yetcites [126] source

In the paper

“Beyond primary sequence coverage, MS/MS analysis has been utilized to study protein complexes [122] and protein-ligand binding [123].”

What the sources say

• Not checked yetcites [122] source
• Not checked yetcites [123] source

In the paper

“Recently, FLASHDeconv used pattern matching to quickly deconvolute MS with speeds orders of magnitudes higher than conventional approaches [128].”

What the sources say

• Not checked yetcites [128] source

In the paper

“For example, Gault et al. demonstrated the use of MS4 analysis to identify both the proteoform state and the associated ligand structure [123].”

What the sources say

• Not checked yetcites [123] source

In the paper

“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].”

What the sources say

• Not checked yetcites [147] source

In the paper

“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].”

What the sources say

• Not checked yetcites [139] source

In the paper

“MS-based proteomics has become a staple in biomedical research for the discovery of novel biomarkers and elucidation of disease mechanisms [140].”

What the sources say

• Not checked yetcites [140] source

In the paper

“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].”

What the sources say

• Not checked yetcites [148] source

In the paper

“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].”

What the sources say

• Not checked yetcites [141] source
• Not checked yetcites [61] source
• Not checked yetcites [142] source
• Not checked yetcites [143] source

In the paper

“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].”

What the sources say

• Not checked yetcites [149] source

In the paper

“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].”

What the sources say

• Not checked yetcites [150] source

In the paper

“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].”

What the sources say

• Not checked yetcites [154] source

In the paper

“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].”

What the sources say

• Not checked yetcites [155] source
• Not checked yetcites [156] source
• Not checked yetcites [157] source

In the paper

“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].”

What the sources say

• Not checked yetcites [81] source
• Not checked yetcites [161] source
• Not checked yetcites [162] source