Nanoproteomics

Enabling Top-Down Proteomics through Nanotechnology and Materials Chemistry

Proteomics is essential for understanding biomolecular functions in cellular systems and their implications in human diseases. A comprehensive analysis of endogenous proteoforms in the human proteome is crucial for uncovering disease mechanisms and identifying therapeutic targets. Native top-down mass spectrometry (MS)-based proteomics enables comprehensive characterization of endogenous proteins and protein complexes, making it the most powerful method for deciphering protein sub-unit assembly and PTM codes alongside sequence variations. Although significant strides have been made recently in both MS hardware and software to advance top-down MS closer to the mainstream, top-down proteomics still faces major challenges. In particular, the complexity of the proteome, coupled with its high dynamic range and the low solubility of numerous proteins, poses significant challenges for high-throughput proteomics studies. In this multidisciplinary project in collaboration with Prof. Ying Ge’s group (https://labs.wisc.edu/gelab/), we will develop novel approaches enabled by nanotechnology and materials chemistry to address these challenges in (native) top-down. Our continuous research will provide innovative tools to enable comprehensive structural characterization of endogenous proteins, protein complexes, macromolecular assembly, non-covalent ligand binding, along with a thorough proteoform landscape of the human proteome. This will significantly advance the understanding of cellular processes and disease mechanisms and identifying potential therapeutic targets to treat human diseases.

Intact Phosphoprotein Analysis by Functionalized Multivalent Nanoparticles

To address the dynamic range of low abundance proteins, we have developed superparamagnetic Fe₃O₄ nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins towards a comprehensive analysis of the phosphoproteome using top-down MS. Subsequently, we developed an integrated top-down phosphoproteomics work flow that coupled NP-based phosphoprotein enrichment by functionalized NPs with online top-down LC/MS/MS to enrich, identify, quantify, and characterize intact phosphoproteins directly from cell lysates and tissue homogenates.

Figure 1. Synthesis of multivalent nanoparticles functionalized with ligand groups to enrich phosphoproteins for the analysis of the phosphoproteome using top-down MS.

Developing a Large-Scale and Reproducible Nanoproteomics Platform

We have further developed a reproducible large-scale synthesis of surface silanized Fe₃O₄ NPs as an enabling nanoproteomics platform and demonstrated its highly specific enrichment of the human heart phosphoproteome. This nanoproteomics platform possesses a unique combination of scalability, specificity, reproducibility, and efficiency for the capture and enrichment of low abundance proteins in general, thereby laying the foundation for practical proteomics applications. We are working on developing novel multivalent nanoparticles functionalized with affinity groups for specific binding to generally capture and enrich other low-abundance proteins of biological or biomedical significance. We will employ these techniques to advance the biological studies and diagnostic assays using such nanotechnology-enabled top-down proteomics.

Figure 2. Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform

Nanoproteomics Enables Proteoform-Resolved Analysis of Cardiac Troponin in Human Serum

We further developed the first proteoform-resolved method for the analysis of low-abundance proteins directly from blood serum enabled by top-down nanoproteomics. We demonstrated this method using cardiac troponin I (cTnI), which is the ‘gold-standard’ biomarker for acute and chronic cardiovascular diseases. Despite their high sensitivity, the current cTnI immunoassays have intrinsic antibody-related limitations and cannot provide the molecular details of cTnI needed for more accurate diagnosis of hearth diseases. We have carefully designed and synthesized peptide-functionalized magnetic Fe₃O₄ NPs using a allene-thiol click chemistry (see Figure below), and demonstrated that they can directly capture and enrich cTnI from human serum (< 1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant blood proteins.

Figure 3. Design, synthesis, and characterization of surface functionalized magnetic nanoparticles (NPs) for capturing cTnI. a, Silanization of Fe₃O₄ NPs using an allene carboxamide-based organosilane monomer (BAPTES) for cysteine thiol-specific bioconjugation. b, Illustration of the rationally designed NPs that are surface functionalized with a 13-mer peptide that has a high affinity for cTnI (NP-Pep) for cTnI enrichment. c-e, Representative TEM images of surface functionalized NPs: Fe₃O₄-OA NPs (c) (inset shows the selected area electron diffraction pattern), Fe₃O₄-BAPTES NPs (d), and Fe₃O₄-Peptide NPs (e).

Antibodies have so far been the dominating affinity reagents for protein capture and quantification in biological research. However, antibody-based platforms suffer from significant limitations including the batch-to-batch variability, high cost of the antibody production, and relatively low chemical stability. In our antibody-free top-down nanoproteomics approach, not only do these NPs outperform conventional monoclonal antibody platforms for serum cTnI enrichment, but also these NPs also faithfully and holistically preserve all endogenous cTnI proteoforms. Thus, these NPs can serve as replacements to conventional immuno-based techniques to overcome the antibody-related limitations in cTnI immunoassays and potentially address the current ‘reproducibility crisis’ caused by antibodies in general. These NPs are highly effective for protein enrichment because: (1) they are commensurate in size and diffusion kinetics with proteins allowing effective penetration through complex biological mixtures; (2) they have high surface-to-volume ratios to enhance protein interaction; (3) they are versatile scaffolds to couple diverse affinity ligands for protein binding and capture. After the specific enrichment by these functionalized NPs, top-down proteomics can reveal diverse cTnI proteoform fingerprints arising from various PTMs of the cTnI directly enriched from blood serum (see Figure below), which can be directly linked to disease phenotypes.

Figure 4. Nanoproteomics enables comprehensive analysis of cTnI proteoforms from human serum. a, Nanoproteomics assay utilizing NP-Pep for specific enrichment of cTnI from serum and subsequent top-down MS analysis of cTnI proteoforms. b, Deconvoluted MS corresponding to cTnI proteoforms enriched from human serum. The cTnI (~10-20 ng/mL) spiked in the human serum (10 mg) were extracted from various human hearts: (i) and (ii), donor hearts; (iii) and (iv), diseased hearts with dilated cardiomyopathy, (v) and (vi), post-mortem hearts.

Structure and Dynamics of Endogenous Cardiac Troponin Complex in Human Heart Tissue Captured by Native Nanoproteomics

Recently, we have further used these functionalized magnetic NPs to enrich and elute endogenous troponin complex directly from heart tissue in its native state, and comprehensively characterized its structure and dynamics by native top-down MS. Native MS analyzes proteins and protein complexes in their native (or near native) state (under non-denaturing solution prior to MS) to preserve their tertiary structure and non-covalent interactions in the gas phase. It has emerged as a powerful tool in structural biology to define protein structure-function relationship. This native nanoproteomics strategy enabled the isotopic resolution of cTn complex, revealing their complex structure and assembly. Further, native MS elucidates the stoichiometry and composition of the cTn complex, localizes Ca2+ binding domains, defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of the proteoform landscape.

Figure. 5. Native nanoproteomics platform for the characterization of endogenous cTn complex from human heart tissue. a Sarcomeric proteins were extracted using a high ionic strength lithium chloride (LiCl) buffer at physiological pH. The heart tissue extract (loading mixture, L) is then incubated with peptide-functionalized nanoparticles (NP-Pep). Following magnetic isolation, the nonspecifically bound proteins are removed as flow through (F). The bound protein complexes are then eluted (E) off the NPs using a native elution buffer containing L-glutamic acid, L-arginine, and imidazole. Native top-down MS (nTDMS) analysis of enriched protein complexes proceeds by either (b) native size exclusion chromatography (SEC) for online buffer exchange and rapid analysis, (c) ultrahigh-resolution Fourier transform ion cyclotron (FTICR)-tandem MS (MS/MS) analysis for probing complex heterogeneity, stoichiometry, and localization of Ca(II) ions, or (d) trapped ion mobility spectrometry (TIMS) coupled with MS (timsTOF) for structural characterization of complex-Ca(II) binding dynamics. e High-resolution mass spectra of endogenous cardiac troponin (cTn) heterotrimeric complexes enriched by NP-Pep directly from human heart tissue and analyzed by nTDMS. f Structural representation showing the cTn heterotrimeric complex. Troponin C (TnC) is depicted in green, cardiac troponin I (cTnI) in purple, cardiac troponin T (cTnT) in orange, and Ca(II) ions in yellow. PDB: 1J1E.

By combining functionalized nanoparticles with top-down mass spectrometry-based proteomics, this approach provides molecular fingerprints of diverse cTnI proteoforms from serum, establishing proteoform-pathophysiology relationships. Native MS further enables protein-complex level characterization from cardiac tissue and can be applied to other low abundance serum proteins and complexes of interest, serving as an enabling technology for top-down proteomics.

Mass Spectrometry-Compatible Surfactants for Improved Protein Solubility and Proteomics Analysis

We also worked with Prof. Ge’s group to design and synthesize photocleavable mass spectrometry compatible surfactants. Protein solubility presents a significant challenge in MS-based proteomics, particularly for membrane proteins. To effectively extract proteins from cells/tissues, surfactants (also known as detergents) are typically employed in the extraction buffer. However, conventional surfactants like SDS (the strongest surfactant) are incompatible with MS due to their higher ionization efficiency, which suppresses the MS signal of proteins. To tackle this issue, we have been developing novel MS-compatible surfactants that degrade quickly into innocuous non-surfactant byproducts, eliminating the need to remove the detergent prior to MS analysis. This collaboration led to the identification of a photo-cleavable anionic surfactant (referred to as “Azo”). Azo can be rapidly degraded upon UV irradiation, enabling the solubilization of membrane proteins and extracellular matrix (ECM) proteins for top-down (as well as bottom-up) proteomics and characterization of PTMs.

Figure 6. A photocleavable surfactant for top-down proteomics.

We further show that this MS‐compatible photocleavable surfactant, 4‐hexylphenylazosulfonate (Azo) can enable a high‐throughput bottom‐up proteomics approach that facilitates robust protein extraction, rapid enzymatic digestion (30 min), and subsequent MS‐analysis following UV degradation. Azo can serve as an “all‐in‐one” MS‐compatible surfactant for both top‐down and bottom‐up proteomics, with streamlined workflows for high‐throughput proteomics amenable to clinical applications.

Figure 7. High throughput proteomics using the “all-in-one” MS compatible photocleavable surfactant Azo.

We have further developed cleavable nonionic surfactants compatible with top-down mass spectrometry.  For example, we synthesized a new family of maltose-derived nonionic surfactants that contain a photocleavable azo-sulfide linker (mAzo), which unfortunately is not very stable in aqueous solution. We have also identified a redox cleavable surfactant, n-Decyl-disulfide-β-D-maltoside (DSSM) suitable for top-down proteomics. We are continuing to develop more nonionic cleavable surfactants that can enable native top-down proteomics.

These innovations have resulted in 5 patents jointly filed by the two groups.

Multidimensional Chromatography for Intact Protein Separation

To address the protein complexity challenges in top-down MS-based proteomics, we have identified ammonium tartrate as a MS-friendly gradient salt, enabling the application of hydrophobic interaction chromatography (HIC), a conventionally MS-incompatible chromatography, to top-down proteomics. We have developed new chromatography materials and novel strategies for multi-dimensional liquid chromatography (MDLC) to separate intact proteins with super high resolution followed by high-resolution MS for protein identification and comprehensive characterization such as novel hydrophobic interaction chromatography (HIC) for native top-down proteomics and serial size exclusion chromatography (sSEC) for large protein MS analysis.

Figure 8. A novel and effective three dimensional liquid chromatography platform coupling ion exchange chromatography, hydrophobic interaction chromatography and reverse phase chromatography for top-down proteomics.

Publications:

28) Chapman EA, Roberts DS, Tiambeng TN, Andrews J, Wang M-D, Reasoner EA, Melby JA, Li BH, Kim D, Alpert AJ, Jin S, Ge Y. Structure and dynamics of endogenous cardiac troponin complex in human heart tissue captured by native nanoproteomics. Nature Commun. 2023, 14, 8400.  doi:10.1038/s41467-023-43321-z. PMCID: PMC10728164

27) Brown KA, Gugger MK, Roberts DS, Moreno D, Chae PS, Ge Y, Jin S. Synthesis, Self-Assembly Properties, and Degradation Characterization of a Nonionic Photocleavable Azo-Sulfide Surfactant Family. Langmuir 2023, 39, 1465-1473. doi: 10.1021/acs.langmuir.2c02820. PMCID: PMC10164600.

26) Brown KA, Gugger MK, Yu Z, Moreno D, Jin S, Ge Y. Nonionic, Cleavable Surfactant for Top-Down Proteomics. Anal Chem. 2023, 95, 1801–1804. doi: 10.1021/acs.analchem.2c03916. PMCID: PMC10323036.

25) Roberts DS, Mann M, Li BH, Kim D, Braiser AR, Jin S, Ge Y. Distinct core glycan and O-glycoform utilization of SARS-CoV-2 Omicron variant Spike protein RBD revealed by top-down mass spectrometry. Chem Sci. 2022 13,10944-10949. doi: 10.1039/d2sc02132c. PMCID: PMC9491206.

24) Buck KM, Roberts DS, Aballo TJ, Inman DR, Jin S, Ponik S, Brown KA, Ge Y. One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant. Anal Chem. 2022, 94, 7164-7168. doi:10.1021/acs.analchem.2c01252. PMCID:PMC9297302.

23)Roberts DS, Mann M, Melby JA, Larson EJ, Zhu Y, Brasier AR, Jin S, Ge Y. Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry. J Am Chem Soc. 2021, 143, 12014-12024. doi: 10.1021/jacs.1c02713. PMCID: PMC8353889.

22) Melby JA, Roberts DS, Larson EJ, Brown KA, Bayne EF, Jin S, Ge Y. Novel Strategies to Address the Challenges in Top-Down Proteomics. J Am Soc Mass Spectrom. 2021, 32,1278-1294. doi: 10.1021/jasms.1c00099. PMCID: PMC8310706.

21) Knott SJ, Brown KA, Josyer H, Carr A, Inman D, Jin S, Friedl A, Ponik SM, Ge Y. Photocleavable Surfactant-Enabled Extracellular Matrix Proteomics. Anal Chem. 2020, 92,15693-15698. doi:10.1021/acs.analchem.0c03104. PMCID:PMC7961849.

20) Brown KA, Tucholski T, Alpert AJ, Eken C, Wesemann L, Kyrvasilis A, Jin S, Ge Y. Top-Down Proteomics of Endogenous Membrane Proteins Enabled by Cloud Point Enrichment and Multidimensional Liquid Chromatography-Mass Spectrometry. Anal Chem. 2020, 92,15726-15735. doi: 10.1021/acs.analchem.0c02533. PMCID: PMC7968110.

19) Timothy N. Tiambeng, David S. Roberts, Kyle A. Brown, Yanlong Zhu, Bifan Chen, Zhijie Wu, Stanford D. Mitchell, Tania M. Guardado-Alvarez, Song Jin & Ying Ge; Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum, Nat. Commun. 2020, 11, 3903.

18) Kyle A. Brown, Trisha Tucholski, Christian Eken, Samantha Knott, Yanlong Zhu, Song Jin, and Ying Ge; High‐Throughput Proteomics Enabled by a Photocleavable Surfactant. Angew. Chem. Int. Ed. 2020, 59 , 8406

17) Kyle A. Brown, Bifan Chen, Tania M. Guardado-Alvarez, Ziqing Lin, Leekyoung Hwang, Serife Ayaz-Guner, Song Jin and Ying Ge; A photocleavable surfactant for top-down proteomics. Nat. Methods, 2019, 16, 417-420. DOI: 10.1038/s41592-019-0391-1. PMCID: PMC6532422.

16) David S. Roberts, Bifan Chen, Timothy N. Tiambeng, Zhijie Wu, Ying Ge and Song Jin; Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform: enrichment of the Human heart phosphoproteome. Nano Res., 2019, 12, 1-9. DOI: 10.1007/s12274-019-2418-4. PMCID: PMC6656398.

15) Trisha Tucholski, Samantha J. Knott, Bifan Chen, Paige Pistono, Ziqing Lin and Ying Ge*; A top-down proteomics platform coupling serial size exclusion chromatography and Fourier transform ion cyclotron resonance mass spectrometry, Anal. Chem. 2019, 91, 3835-3844. DOI: 10.1021/acs.analchem.8b04082. PMCID: PMC6545233

14) Yu Liang, Yutong Jin, Zhijie Wu, Trisha Tucholski, Kyle A. Brown, Lihua Zhang*, Yukui Zhang and Ying Ge*; Bridged Hybrid Monolithic Column Coupled to High-Resolution Mass Spectrometry for Top-down Proteomics, Anal. Chem. 2019, 91, 1743-1747. DOI: 10.1021/acs.analchem.8b05817. PMCID: PMC6491350.

13) Bifan Chen, Ziqing Lin, Andrew J. Alpert, Cexiong Fu, Qunying Zhang, Wayne A. Pritts and Ying Ge*; Online hydrophobic interaction chromatography-mass spectrometry for the analysis of intact monoclonal antibodies, Anal. Chem. 2018, 90, 7135-7138. DOI: 10.1021/acs.analchem.8b01865. PMCID: PMC6109971.

12) Zhijie Wu, Timothy N. Tiambeng, Wenxuan Cai, Bifan Chen, Ziqing Lin, Zachery R. Gregorich and Ying Ge*; Impact of phosphorylation on the mass spectrometry quantification of intact phosphoproteins, Anal. Chem. 2018, 90, 4935-4939. DOI:10.1021/acs.analchem.7b05246. PMCID: PMC6138620.

11) Bifan Chen, Kyle A. Brown, Ziqing Lin and Ying Ge*; Top-down proteomics: ready for prime time? Anal. Chem. 2018, 90, 110-127. DOI: 10.1021/acs.analchem.7b04747. PMCID: PMC6138622.

10) Wenxuan Cai, Trisha Tucholski, Bifan Chen, Andrew J. Alpert, Sean McIlwain, Takushi Kohmoto, Song Jin, and Ying Ge; Top–down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy, Anal. Chem. 2017, 89, 5467-5475, DOI: 10.1021/acs.analchem.7b00380

9) Bifan Chen, Leekyoung Hwang, William Ochowicz, Ziqing Lin , Tania Maria Guardado-Alvarez, Wenxuan Cai, Lichen Xiu, Kunal Dani, Cyrus Colah, Song Jin and Ying Ge; Coupling Functionalized Cobalt Ferrite Nanoparticle Enrichment with Online LC/MS/MS for Top-down Phosphoproteomics, Chem. Sci., 2017, 8, 4306-4311, DOI: 10.1039/C6SC05435H

8) Leekyoung Hwang, Tania M. Guardado-Alvarez, Serife Ayaz-Gunner, Ying Ge, and Song Jin; A Family of Photolabile Nitroveratryl-Based Surfactants That Self-Assemble into Photodegradable Supramolecular Structures, Langmuir, 2016, 32, 3963–3969, DOI: 10.1021/acs.langmuir.6b00658

7) Bifan Chen, Ying Peng, Santosh G. Valeja, Lichen Xiu, Andrew J. Alpert, and Ying Ge*; Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics, Anal. Chem., 2016, 88, 1885–1891, DOI: 10.1021/acs.analchem.5b04285

6) Santosh G. Valeja, Lichen Xiu, Zachery R. Gregorich, Huseyin Guner, Song Jin, and Ying Ge; Three Dimensional Liquid Chromatography Coupling IEC/HIC/RPC for Effective Protein Separation in Top-Down Proteomics, Anal. Chem., 2015, 87, 5363–5371, DOI: 10.1021/acs.analchem.5b00657

5) Ying-Hua Chang, Zachery r. Gregorich, Albert J. chen, Leekyoung Hwang, Huseyin Guner, Dyang Yu, Jianyi, and Ying Ge*; New Mass-Spectrometry-Compatible Degradable surfactant for Tissue Proteomics, Journal of Proteome Res. 2015, 14, 1587–1599 DOI: 10.1021/pr5012679

4) Leekyoung Hwang, Serife Ayaz-Guner, Zachery R. Gregorich, Wenxuan Cai, Santosh G. Valeja, Song Jin, and Ying Ge; Specific Enrichment of Phosphoproteins Using Functionalized Multivalent Nanoparticles, J. Am. Chem. Soc., 2015, 137, 2432-2435, DOI: 10.1021/ja511833y

3) Lichen Xiu, Santosh G. Valega, Andrew J. Alpert, Song Jin, and Ying Ge; Effective Protein Separation by Coupling Hydrophobic Interaction and Reverse Phase Chromatography for Top-down Proteomics, Anal. Chem., 2014, 86, 7899-7906, DOI: 10.1021/ac501836k

2) Nelson, C. A.; Szezech, J. R.; Zhu, H.; Xu, Q.; Lawrence, M. J.; Jin, S.; Ge, Y.; Effective Enrichment and Mass Spectrometry Analysis of Phosphopeptides Using Mesoporous Metal Oxide Nanomaterials, Anal. Chem. 2010, 82, 7193-7201.

1) Nelson, C. A.; Szczech, J. R.; Xu, Q.; Lawrence, M. J., Jin, S.; Ge, Y.; Mesoporous Metal Oxide Nanomaterials Effectively Enrich Phosphopeptides for Mass Spectrometry-based Phosphoproteomics, Chem. Commun. (2009) 6607-6609

Patents:

7) Ge, Y.; Jin, S.; Brown, K.; Gugger, M.; “A Nonionic, Redox-Cleavable Surfactant for Mass Spectrometry-Based Proteomics and Structural Biological Applications” Provisional Patent filed August 17, 2023. 18/451614

6) Ge, Y.; Jin, S.; Brown, K.; Gugger, M.; “A Nonionic, Redox-Cleavable Surfactant for Mass Spectrometry-Based Proteomics” Provisional Patent filed August 18, 2022.

5) Ge, Y.; Jin, S.; Brown, K.; Buck, K.; “A Novel Strategy Enabled by a Photo-cleavable Surfactant for Extracellular Vesicle Proteomics” Provisional Patent filed Nov 2, 2021; US patent filed Nov 1, 2022.

4) Ge, Y.; Jin, S.; Tiambeng, T. N.; Roberts, D. S.; “An Accurate and Comprehensive Cardiac Troponin I Assay Enabled by Nanotechnology and Proteomics” Provisional Patent 62/949896, filed Dec 18, 2019. PCT patent PCT/US20/66011 filed Dec 18, 2020.

3) Ge, Y.; Jin, S.; Guardado-Alvarez, T.; Brown, K.; “Photo-cleavable Surfactants for Top-Down Proteomics” Provisional Patent 62/682,027 filed June 7, 2018; PCT Patent “Photo-cleavable Surfactants” PCT/US19/35447 filed June 4, 2019, Issued Sept 2022. US Patent US 11,567,085 B2 issued Jan 31, 2023. Licensed to Sigma-Aldrich Sept 2020.

2) Ge, Y.; Jin, S.; Guardado-Alvarez, T.; Brown, K.; “Photo-cleavable Surfactants for Top-Down Proteomics” Provisional Patent 62/682,027 filed June 7, 2018; Second Provisional patent filed 62/810,744 filed February 26, 2019; PCT Patent ““Photo-cleavable Surfactants“ filed May 30, 2019.

1) Jin, S; Ge, Y; Nelson, C, Xu, Q; Use of nanomaterials to Enrich Phosphopeptides for Ma ss Spectrometry-Based P roteomics, 2013