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

In the paper

“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].”

What the sources say

• Supported

  The opening section of “Proteoforms as the next proteomics currency” (page not provided) defines proteoforms as forms arising from sequence variations, splice isoforms, and posttranslational modifications. It also describes these sources of variation as creating a complex diversity of proteins, supporting the citing sentence’s paraphrase. (LLM, conf=0.91)

  cites [1] source
• Not checked yetcites [2] source
• Supported

  In “To the Editor,” the paper says protein complexity arises from allelic variation, alternative RNA splicing, and post-translational modifications. It defines proteoforms as the different molecular forms of a single gene’s protein product, including forms arising from genetic variation, splicing, and PTMs. (LLM, conf=0.94)

  cites [3] source
• Supported

  Figure 2 (p. 20) says that alternatively spliced isoforms and site-specific changes, including SNPs and PTMs, combine to generate proteoforms that map back to a single gene. Figure 3 (p. 24) also identifies mutations, alternative splicing, and PTMs as sources of protein variability. (LLM, conf=0.94)

  cites [4] source

In the paper

“Liang et al. demonstrated that bridged hybrid bis(triethoxysilyl)ethylene monolith with C8 functional groups afforded high-resolution separation of complex cardiac lysate including a large proteoform, α-actinin2 (103.77 kDa) [72].”

What the sources say

• Supported

  The Abstract identifies the column as a bridged hybrid bis(triethoxysilyl)ethylene monolith with C8 functional groups. It reports effective separation of a complex protein mixture from swine cardiac tissue and identification of α-actinin2 (103.77 kDa) among the proteoforms. (LLM, conf=0.94)

  cites [72] 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

• Partly supported

  The Conclusions and Future Directions section says that important molecular mechanisms remain unresolved, including how sarcomere changes cause cell enlargement and fibrosis. That supports the quoted point about the HCM phenotype. The same citation is attached to the full sentence, however, and the Molecular Genetics section reports more than 1,000 rare pathogenic variants in HCM and DCM, not the stated figure of around 1,400 mutations in 11 sarcomere genes; those exact numbers are not supported by our copy. (LLM, conf=0.85)

  cites [148] sourceExperimental — accuracy not yet measured

In the paper

“Undoubtedly, the advancement of top-down proteomics is stimulated by the commercial growth of large molecule therapeutics such as monoclonal antibodies and antibody-drug-conjugates as detailed and high-throughput methods for their characterization and quality control are required [81,159,160].”

What the sources say

• Partly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [81] sourceExperimental — accuracy not yet measured
• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [159] sourceExperimental — accuracy not yet measured
• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [160] sourceExperimental — accuracy not yet measured

In the paper

“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].”

What the sources say

• Not checked yetcites [13] source
• Not checked yetcites [14] source

In the paper

“To standardize sample preparation, a “best practices and benchmarks” was reported by Agar and colleagues in the Consortium of Top-down Proteomics [23].”

What the sources say

• Not checked yetcites [23] source

In the paper

“Mass spectrometry (MS)-based proteomics has emerged as the most versatile and comprehensive strategy for protein characterization [8,9].”

What the sources say

• Not checked yetcites [8] source
• Not checked yetcites [9] source

In the paper

“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].”

What the sources say

• Not checked yetcites [5] source
• Not checked yetcites [7] source

In the paper

“However, most salts and conventional surfactants are generally incompatible with MS analysis and must be removed before downstream analysis [22].”

What the sources say

• Not checked yetcites [22] source

In the paper

“However, it was found that these surfactants are incompatible with top-down proteomics [31].”

What the sources say

• Not checked yetcites [31] source

In the paper

“Previously, acid-cleavable surfactants such as Rapigest [28], ProteaseMAX [29], and MS-compatible degradable surfactant MaSDeS [30] were developed as MS-compatible surfactants to enable protein solubilization for digestion and MS-analysis.”

What the sources say

• Not checked yetcites [28] source
• Not checked yetcites [29] source
• Not checked yetcites [30] source

In the paper

“To improve the sensitivity and automation of top-down sample preparation, Zhu and coworkers have adapted the nanoPOTS (nanodroplet processing in one pot for trace samples) [37] technology for top-down proteomics.”

What the sources say

• Not checked yetcites [37] source

In the paper

“Azo was used to broadly solubilized proteins from cardiac tissues including important membrane proteins such as phospholamban [32], receptor expression enhancing protein 5 [33], and every subunit of the ATP synthase complex [34,35].”

What the sources say

• Not checked yetcites [32] source
• Not checked yetcites [33] source
• Not checked yetcites [34] source
• Not checked yetcites [35] He J, 2018, Assembly of the membrane domain of ATP synthase in human mitochondria. Proceedings of the National Academy of Sciences

In the paper

“In particular, precipitation is often required when using surfactants, which are often employed during cell lysis to solubilize hydrophobic membrane proteins, because they significantly inhibit MS signal by outcompeting proteins for ionization or by forming harmful adducts [24,25].”

What the sources say

• Not checked yetcites [24] source
• Not checked yetcites [25] source

In the paper

“To address this gap in technology development, the Ge group has developed superparamagnetic cobalt ferrite nanoparticles (NPs) functionalized with GAPT-Zn ligands for phosphoprotein enrichment before LC-MS/MS analysis for confident identification and localization of phosphorylation sites [57].”

What the sources say

• Not checked yetcites [57] source

In the paper

“Robinson and coworkers have successfully demonstrated native MS analysis for a number of applications including most recently analyzing protein assemblies directly for native membranes bypassing the use of detergents [51].”

What the sources say

• Not checked yetcites [51] source

In the paper

“The human proteome has a high dynamic range; therefore, the analysis of certain protein classes, low abundance proteoforms, and target proteins often requires an enrichment step [4,52].”

What the sources say

• Not checked yetcites [4] source
• Not checked yetcites [52] Anderson NL, 2002, History Charact Diagn Prospects

In the paper

“Due to the hydrophobic nature of membrane proteins, the use of nonionic surfactants, such as DDM or the recently developed oligoglycerol class of detergents (shown to improve purification and MS-based characterization of G-protein coupled receptors) [47], to solubilize and stabilize membrane proteins is generally required to enable native MS analysis [48–50].”

What the sources say

• Not checked yetcites [47] source
• Not checked yetcites [48] source
• Not checked yetcites [49] source
• Not checked yetcites [50] source

In the paper

“They found that a combination of dodecyl-β-D-maltopyranoside (DDM) with urea enabled the extraction and identification of ~170–620 proteoforms from as little as ~70–770 HeLa cells [38].”

What the sources say

• Not checked yetcites [38] source

In the paper

“On the other hand, intact proteoform enrichment, which is necessary for top-down proteomics, is relatively underdeveloped [56].”

What the sources say

• Not checked yetcites [56] source

In the paper

“Overall, the NP-peptide approach outperformed conventional monoclonal antibody and top-down characterization of cTnI, enriched from serum, yielded proteoform-resolved molecular fingerprints of diverse cTnI proteoforms for evaluation of proteoform-pathophysiology relationships (Figure 3) [68].”

What the sources say

• Not checked yetcites [68] source

In the paper

“To address the limitation of the antibody-based approach, Tiambeng and Roberts led the development of a nanoproteomics strategy using functionalized magnetic NPs with a peptide affinity ligand to specifically target cardiac troponin I (cTnI) [65–67], an important and gold-standard cardiac biomarker [68].”

What the sources say

• Not checked yetcites [65] source
• Not checked yetcites [66] source
• Not checked yetcites [67] source

In the paper

“Overall, the NP-peptide approach outperformed conventional monoclonal antibody and top-down characterization of cTnI, enriched from serum, yielded proteoform-resolved molecular fingerprints of diverse cTnI proteoforms for evaluation of proteoform-pathophysiology relationships (Figure 3) [68].”

What the sources say

• Not checked yetcites [68] source

In the paper

“The newly developed surface-functionalized NP platform could be further generalized to other enrichment targets, provided a suitable affinity reagent is available [58].”

What the sources say

• Not checked yetcites [58] source

In the paper

“However, front-end separation of intact proteins represents a major challenge for in-depth proteome coverage in part because of dramatic variation in physicochemical properties of proteins (size, hydrophobicity, charge, etc.).”

In the paper

“The development of sheathflow and sheathless interfaces have enabled facile coupling of CZE to MS [73].”

What the sources say

• Not checked yetcites [73] source

In the paper

“Capillary zone electrophoresis (CZE) has recently shown great promise as an alternative or complementary front-end separation due to its inherent sensitivity and peak capacity [73].”

What the sources say

• Not checked yetcites [73] source

In the paper

“However, the application of CZE for intact protein separation has been limited by the low loading capacity and narrow separation window common to CZE systems.”

In the paper

“While the continued development of online separation strategies is essential for enhancing the capabilities of top-down proteomics, no single separation mode can sufficiently handle the complexity of the whole proteome. Therefore, multidimensional separation approaches are commonly employed for deeper proteome coverage [82].”

What the sources say

• Not checked yetcites [82] source

In the paper

“Hydrophobic interaction chromatography (HIC)-MS shows great promise for characterizing intact protein directly coupled to top-down MS [80].”

What the sources say

• Not checked yetcites [80] source

In the paper

“SEC was also coupled to CZE for the identification of ~5700 proteoforms from Escherichia coli [86].”

What the sources say

• Not checked yetcites [86] source

In the paper

“However, some recent online approaches have been developed which include using weak-cationic exchange HILIC and RPLC to enable the characterization of hundreds of histone proteoforms from ~1.5 μg of protein [90].”

What the sources say

• Not checked yetcites [90] source

In the paper

“In 2012, the Kelleher group combined solution isoelectric focusing, GELFrEE, and RPLC-MS/MS to identify ~3,000 proteoforms from 1043 protein groups including proteins up to 105 kDa [84].”

What the sources say

• Not checked yetcites [84] source

In the paper

“Using Native GELFrEE and IEX, Skinner et al. identified 125 intact endogenous complexes from mouse hearts and human cancer cell lines [89].”

What the sources say

• Not checked yetcites [89] source

In the paper

“Overall, gel-based strategies suffer from the use of SDS, which requires sample clean-up that may result in protein loss and variability [26].”

What the sources say

• Not checked yetcites [26] source

In the paper

“Yu et al. took a different approach using offline fractionation by high pH RPLC followed by online low pH RPLC coupled to MS to enable the identification of 2778 proteoforms from 628 proteins [87].”

What the sources say

• Not checked yetcites [87] source

In the paper

“Recently, submicron ESI emitter tips, developed by the Williams group, have pushed the boundaries of ESI sensitivity while also showing improved salt tolerance up to physiological concentrations (25 mM Tris, 150 mM KCl) by virtue of creating smaller droplets [93].”

What the sources say

• Not checked yetcites [93] source

In the paper

“Although ESI enables the highly sensitive detection of analytes, protein detection by ESI suffers from signal competition with small-molecule contaminates that outcompete large molecules and results in the need for highly purified samples [23,83].”

What the sources say

• Not checked yetcites [23] source
• Not checked yetcites [83] source

In the paper

“However, top-down proteomics using an Orbitrap MS was limited to low-molecular-weight species as the signal of larger biomolecules rapidly decays due to collisions with the background gas [101].”

What the sources say

• Not checked yetcites [101] source

In the paper

“Additionally, online high pH RPLC coupled to low pH RPLC shows promise for intact protein separation before top-down MS [91].”

What the sources say

• Not checked yetcites [91] source

In the paper

“Moreover, global top-down proteomics using the Orbitrap has enabled the identification and quantification of thousands of proteoforms [84,100].”

What the sources say

• Not checked yetcites [84] source
• Not checked yetcites [100] source

In the paper

“Work by the Brodbelt and Kelleher group in tuning the Orbitrap instrument parameters has increased its scope for targeted and global analysis of larger proteoforms [98,102].”

What the sources say

• Not checked yetcites [98] source
• Not checked yetcites [102] source

In the paper

“More recently, instrument hardware improvements have led to enhanced large ion transmission and detection, especially with the implementation of ultra-high mass range (UHMR) Orbitraps that are capable of analyzing species at 70,000 m/z and mass as large as 800 kDa [103,104].”

What the sources say

• Not checked yetcites [103] source
• Not checked yetcites [104] source

In the paper

“CDMS analysis of intact proteins is achieved by simultaneously measuring m/z and z for individual ions contained in an electrostatic ion trap from the frequency and amplitude of the resulting signal, which can then be converted into a mass measurement [111].”

What the sources say

• Not checked yetcites [111] source

In the paper

“Arguably, one of the most important recent developments is charge detection mass spectrometry (CDMS) (also referred to as individual ion MS) for top-down proteomics [107–110].”

What the sources say

• Not checked yetcites [107] source
• Not checked yetcites [108] source
• Not checked yetcites [109] source
• Not checked yetcites [110] source

In the paper

“The simplest of these is using non-denaturing conditions, which inherently improves the detection of larger biomolecules by reducing the number of protein charge states to collapse MS signal into fewer isotopic features [105].”

What the sources say

• Not checked yetcites [105] source

In the paper

“Effective ion activation for MS/MS analysis is essential for characterizing proteoforms [112].”

What the sources say

• Not checked yetcites [112] source

In the paper

“Collisionally activated dissociation (CAD) and higher-energy collisional dissociation (HCD) are the most prevalent and robust techniques for protein fragmentation especially for global top-down proteomics studies [100,113].”

What the sources say

• Not checked yetcites [100] source
• Not checked yetcites [113] source

In the paper

“Electron capture dissociation (ECD) [114] and electron transfer dissociation (ETD) [115], on the other hand, are nonergodic techniques for protein fragmentation.”

What the sources say

• Not checked yetcites [114] source
• Not checked yetcites [115] source

In the paper

“Recently, combining deconvolution results from a single data set using a simple voting ensemble method or random forest machine learning-enabled improved fragment identification and confidence compared to a single algorithm [131].”

What the sources say

• Not checked yetcites [131] source

In the paper

“Using the ‘Nativeomics’ approach, endogenous ligands such as lipids and even drug binding could be identified as demonstrated for several membrane proteins.”

What the sources say

• Not checked yetcites [123] source

In the paper

“Importantly, AI-ETD generally outperformed HCD, ETD, and EThCD in terms of sequence coverage and proteoform characterization scores.”

What the sources say

• Not checked yetcites [121] source

In the paper

“Additionally, decreased level of cTnI phosphorylation was found in hearts with increased chronic heart failure [61]”

What the sources say

• Not checked yetcites [61] source

In the paper

“Their results convincingly show that regardless of the HCM causing mutation, convergent sarcomeric proteoforms were observed in the HCM samples compared to the donor samples.”

What the sources say

• Not checked yetcites [149] source

In the paper

“Notably, the use of top-down proteomics to identify characteristic PTM or isoform profiles from low-abundance proteins shows promise as a “molecular fingerprint” with the potential to serve as the next-generation biomarkers.”

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

“Using the previously discussed NP-based method, Tiambeng and Roberts et al. demonstrated that different low-abundance cTnI proteoforms could be enriched directly from human serum and characterized by top-down MS, showing promise for improved clinical diagnosis [68].”

What the sources say

• Not checked yetcites [68] source

In the paper

“Tucholski et al. performed a large-scale quantitative top-down proteomics study of donor (n=16) and obstructive HCM (n=16) samples to compare the proteoform landscape between the two groups (Figure 4) [149].”

What the sources say

• Not checked yetcites [149] source

In the paper

“Wei et. al applied a top-down proteomics strategy to study the sarcomere proteome of fast- and slow-twitch skeletal muscles of aging rats [153].”

What the sources say

• Not checked yetcites [153] source

In the paper

“This finding represented the first report of PTMs in Z-disk proteins and implies the importance of the Z-disk in the progression of sarcopenia.”

What the sources say

• Not checked yetcites [153] source

In the paper

“In addition, Tucholski et al. also observed highly consistent changes in the phosphorylation levels of cardiac troponin T, tropomyosin isoform 1.1, and the ventricular isoform of myosin light chain 2 in the HCM samples.”

What the sources say

• Not checked yetcites [149] source

In the paper

“Distinct changes in protein isoform expression, such as a switch in expression from the atrial to the ventricular isoform of myosin light chain 1, were observed between the two maturation states.”

What the sources say

• Not checked yetcites [151] source

In the paper

“This study indicates a conserved pathway is associated with clinical phenotypes in patients diagnosed with obstructive HCM, providing a new opportunity for the development of interventions that target the HCM phenotype rather than the individual genotype [149].”

What the sources say

• Not checked yetcites [149] source

In the paper

“While this report was important to study the effects of sarcopenia, the study was limited to only two skeletal muscle tissues: gastrocnemius (fast-twitch) and soleus (slow-twitch).”

What the sources say

• Not checked yetcites [153] source

In the paper

“This finding represented the first report of PTMs in Z-disk proteins and implies the importance of the Z-disk in the progression of sarcopenia.”

What the sources say

• Not checked yetcites [153] source

In the paper

“The top-down proteomics was employed to quantify contractile protein isoform and PTM changes between early and late-stage 2D hiPSC-CM monolayers and 3D hiPSC-ECTs (Figure 5) [151].”

What the sources say

• Not checked yetcites [151] source

In the paper

“For the first time, contractile PTMs such as a decrease in alpha tropomyosin phosphorylation were discovered as promising new markers of hPSC-CMs maturation.”

What the sources say

• Not checked yetcites [151] source

In the paper

“To address this challenge, Cai et al. developed an unbiased method for the assessment of hiPSC-CMs maturation by combining functional assessments and proteomics to assess CM maturation in 2-dimensional (2D) monolayer systems and 3-dimensional (3D) engineered cardiac tissues (ECTs) [151].”

What the sources say

• Not checked yetcites [151] source

In the paper

“Melby et al. expanded upon this study by performing top-down proteomics on seven different rat skeletal muscle tissues: vastus lateralis (VL), vastus medialis (VM), vastus intermedius (VI), rectus femoris (RF), soleus (SOL), gastrocnemius (GAS), and plantaris (PLN) [154].”

What the sources say

• Not checked yetcites [154] source

In the paper

“Importantly, quantitative top-down proteomics revealed age-related alterations, such as the phosphorylation levels of the Z-disk protein cypher, in the fast-twitch skeletal muscles of the aging rats [153].”

What the sources say

• Not checked yetcites [153] 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

“As the number of large-molecule therapeutics increases, top-down proteomics is expected to be increasingly utilized by the pharmaceutical industry.”

In the paper

“They identified changes in PTMs (such as Cys118 nitrosylation and C-terminal methylation) using both isogenic KRAS colorectal cancer (CRC) cell lines and patient CRC tumors.”

What the sources say

• Not checked yetcites [59] source

In the paper

“Targeted top-down proteomics was employed by Ntai et al. to investigate mutation in KRAS [59].”

What the sources say

• Not checked yetcites [59] source

In the paper

“He et al. utilized ultrahigh-resolution MS on a 21 T FTICR enabling the diagnosis of hemoglobinopathy and β-thalassemia from blood with a 3 min assay [158].”

What the sources say

• Not checked yetcites [158] source

In the paper

“Recently, Toby et al. developed a top-down proteomics-based protocol for the analysis of human peripheral blood mononuclear cells [157].”

What the sources say

• Not checked yetcites [157] source