Enabling Top-Down Proteomics through Nanotechnology and Materials Chemistry
Proteomics is essential for deciphering how biomolecules interact as a system and for understanding the functions of cellular systems in human diseases. A comprehensive analysis of all proteoforms in the human proteome is essential for gaining a transformative understanding of disease mechanisms and identifying new therapeutic targets. Top-down mass spectrometry (MS)-based proteomics analyzes intact proteins and is the most powerful method to comprehensively characterize proteoforms to decipher the PTM codes together with 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 proteome is extremely complex and has a high dynamic range in addition to the low solubility of many proteins, making it highly challenging for high-throughput proteomic study. 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 top-down MS-based proteomics.
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.
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.
By using functionalized nanoparticles in combination with top-down mass spectrometry-based proteomics, this nanoproteomics approach provides proteoform-resolved molecular fingerprints of diverse cTnI proteoforms from serum to establish proteoform-pathophysiology relationships. Beyond cTnI, we expect this nanoproteomics approach can be generally applied to other low abundance serum proteins of interest and can serve as an enabling technology for top-down serum proteomics.
Mass Spectrometry-Compatible Surfactants for Improved Protein Solubility and Proteomics Analysis
Protein solubility is a significant challenge in MS-based proteomics, especially for membrane proteins. To effectively extract proteins from cells/tissues, surfactants (also known as detergents) are typically used in the extraction buffer. However, conventional surfactants such as SDS (the strongest surfactant) are not compatible with MS due to their higher ionization efficiency which suppress the MS signal of proteins. To address this problem, we have been developing novel MS-compatible surfactants that can quickly degrade into innocuous non-surfactant byproducts, eliminating the need to remove the detergent prior to MS analysis. Particularly, we have successfully identified a photo-cleavable anionic surfactant, 4-hexylphenylazosulfonate (referred to as Azo), that can effectively solubilize proteins with similar performance to SDS and be rapidly degraded upon UV irradiation for top-down proteomics. Importantly, Azo-aided top-down proteomics enables the solubilization of membrane proteins for comprehensive characterization of PTMs.
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.
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 7. A novel and effective three dimensional liquid chromatography platform coupling ion exchange chromatography, hydrophobic interaction chromatography and reverse phase chromatography for top-down proteomics.
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
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 Mass Spectrometry-Based Proteomics, 2013