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Home> Events> Professor Lingjun Li’s group published two papers in Nature Communications
Professor Lingjun Li’s group published two papers in Nature Communications
Publish Date:2020/05/18   |   Click:1018

 Li Research Group  University of Wisconsin-Madison

Lingjun Li Research Group (LRG)

Li Research Group (LRG) is a bioanalytical chemistry laboratory at the School of Pharmacy and Department of Chemistry, University of Wisconsin-Madison, established in 2003, and successfully trained 48 PhD scientists and a dozen postdocs to date. It currently consists of 22 PhD graduate students, 6 postdoctoral scientists and 7 undergraduate researchers. Under the leadership of Professor Lingjun Li, the LRG has been focusing on the development and application of an array of novel mass spectrometry (MS)-based tools and strategies, to study challenging neuroscience and biomedical problems. Using a multi-faceted platform combining chemical labeling, micro-scale separation (capillary electrophoresis and nanoLC), and tandem MS sequencing techniques, the LRG discovered more than 300 novel neuropeptides in crustacean model organisms in the past decade. Furthermore, both mass spectrometric imaging technology and in vivo microdialysis sampling tools have been implemented to follow neuropeptide distribution and secretion with unprecedented details.  Additionally, the LRG also explores novel use of ion mobility MS to address technical challenges in peptidomic research, including site-specific peptide epimer analysis and improvement of isobaric tandem MS quantitation. The cutting-edge analytical tool development enables large-scale proteomics and posttranslational modification analyses and their applications in biomarker discovery and clinical translational research efforts in Alzheimer’s disease, autism spectrum disorder, diabetes, pancreatic cancer, and breast cancer, among others. In conjunction with their efforts in proteomics and metabolomics, the LRG has also been actively developing several innovative quantitative schemes and new isobaric tagging reagents based on dimethylated amino acids (e.g., Dimethylated leucine (DiLeu) isobaric tags), DiPyrO tags and SUGAR tags that enable high-throughput quantitative proteomics and glycomics studies. These synergistic projects span analytical mass spectrometry, capillary separations, peptide chemistry, neurochemistry, and neurobiology. The improved analytical method development enables biological discovery, and the emerging biological questions require further advancement of analytical tools.  We highlight below two recent studies from the LRG published in Nature Communications.

LRG Research Highlight 1--- New insights into Alzheimer’s disease revealed by IMS

With the current lack of effective treatment of Alzheimer’s disease (AD), there is an urgent need for earlier, possibly preventative intervention, raising the question of what form of amyloid beta (Aβ) is the best target. Chiral inversion of amino acids is thought to modulate the structure and function of many neuropeptides, including Aβ, but these processes are poorly understood. As chiral Aβ peptides with partial amino acid D-isomerization have been previously detected in AD brain regions, there is a possibility that D-isomerized Aβ play a vital role in AD pathogenesis and development. However, since Aβ D-isomerization is age-dependent and is present at low stoichiometry (e.g. less than 10%), the role of chiral Aβ has long been ignored and largely underexplored, in part due to lack of effective tools. To address this knowledge gap, the LRG proposes a novel concept and potentially new drug target for AD therapy by investigating chiral effects on Aβ peptide. In the recent paper (Nat. Commun.2019, 10, 5038) published in Nature Communications, LRG reported an integrated ion mobility-mass spectrometry (IM-MS)-based approach to study chirality-regulated structural features of Aβ fragments and their influence on Aβ receptor recognition.

Based on their accumulated IM-MS research experience (J. Am. Soc. Mass Spectrom. 2017, 28, 110/Anal. Chem. 2014, 86, 2972/Trends in Anal. Chem. 2019, in press, doi: 10.1016/j.trac.2019.05.048), the LRG developed and established an innovative analytical platform, benefiting from the rational designs that target Aβ chiral chemistry. Consequently, distinct structural and molecular differences have been revealed between wild type and D-isomerized Aβ, including its monomer structure, oligomerization behavior and its receptor-recognition and binding characteristics. In addition to the crosstalk effects among those epimeric Aβ during oligomerization, the differential contributions of the chirality of Aβ N-terminal and C-terminal fragments were also interrogated, suggesting their inevitably cooperative effects. It is believed that current results could facilitate future investigations of novel therapeutic treatments for AD as new insights can be obtained via elucidation of the roles of D-isomerized Aβ in early AD development, diagnosis, and prognosis. Notably, this study highlighted the importance of considering key amino acid chirality in Aβ, when designing inhibitors that target to disrupt or direct stereoisomeric Aβ to stable, nontoxic and off-pathway aggregation. 

To rationally design such stereoisomeric Aβ inhibitors, it is urgently required to better understand the differences between stereoisomeric Aβ and wild type counterparts, including monomer structure, aggregation pathway and receptor recognition behavior. The key idea of chiral amplification through metal binding was originally inspired by previous reports including our own research experience on zinc finger peptide-zinc binding and other IM-MS-based peptide-metal binding studies. The first author of the paper, Dr. Gongyu Li, decided to choose copper as the candidate metal, as it has unique isotopic distribution, binds to most peptides with a moderate to high affinity, allowing the binding events to be captured in the gas phase even after desolvation, as well as its biomedical relevance. The next question, however, was concerning the strategy to maximize the chiral amplification power and how to quantitatively characterize/report such chiral amplification. Gongyu adopted a previous multidimensional data visualization method, but with significant modifications, to include more rational choice of individual coordinates/vectors, namely, the collisional cross-sections (CCS) for zero-, one- and two-copper-bound peptide species. These three CCSs are highly dependent on the metal-binding events and thus the chiral effects can be evaluated. The next step was to plot each of these 3D vectors into a 3D scattering space. By defining the spatial distance in this 3D scattering space to quantitatively characterize the D/L Structural Difference (DLSD), this method allows evaluation of the Aβ oligomer chiral amplification in a quantitative manner. Collectively, the creation of this IM-MS-based integrative chirality anatomy platform (iCAP) will enable more in-depth investigation of previously ignored peptide and protein structural differences caused by amino acid chirality. To read the full article, please click here (https://www.nature.com/articles/s41467-019-12346-8).

LRG Research Highlight 2--- Nanosecond photochemical reaction coupled with MS

In another recent publication from the LRG (Nat. Commun.2019, 10, 4697, featured by Nature Communications Editors Highlights) also led by postdoc Gongyu Li, published in Nature Communications, the team demonstrated the suitability and possibility to adapt nanosecond photochemical reaction (nsPCR) with mass spectrometry (MS) for multiple analytical measurements and biological applications. This development has enabled sample separation, MS ionization, and chemical reaction to be integrated into a localized space.

Simultaneous protein identifications and their structural probing are frequently beset with tedious, multistep and structural perturbing sample preparations. The resultant relatively independent workflows for each other have thus discouraged their applications in rapid and reliable probing of biological and medical events. In this work, Li and coworkers presented a “Two-Birds-With-One-Stone” photochemical strategy for a trade-off between large protein structural probing and on-demand matrix removal. The nsPCR is designed to enable simultaneous high-throughput, highly efficient protein labeling and on-demand matrix removal at nanosecond timescale and micrometer confined environment. The labeling occurs between the nsPCR product, 2-nitrosobenzoic anion (NS-), and primary amine groups at the N-terminal and lysine residues of proteins. The on-demand matrix removal benefits from the composite effects of local pH-jump effect, photochemical microscale electrophoresis and microscale thermophoresis. The labeling efficiency for various sample types has been demonstrated to be as high as 90% in a high-throughput manner. As a proof-of-concept demonstration, the nsPCR has also revealed the stabilizing effects of terminal sialylation on glycoprotein 3D structures for the first time, which offers unique features and capabilities that cannot be readily achieved using a typical gas-phase structural analysis tool, ion mobility-mass spectrometry.

The key idea of nsPCR stemmed from previous use of photoactive compound 2-nitrobenzaldehyde (NBA), which releases a proton upon UV laser irradiation (J. Am. Chem. Soc. 2016, 138, 5363), and generates amine reactive species. Dr. Gongyu Li, a postdoc from the LRG, and also the first author of two recent Nature Communications papers (Nat. Commun.2019, 10, 4697/Nat. Commun.2019, 10, 5038), began to explore its utility in matrix-assisted desorption ionization (MALDI)-MS in terms of alleviating ion suppression effects. Notably, the LRG has been long interested in improving methods for in situ identification and visualization of neuropeptides and proteins directly from multiple tissue samples, which is often plagued by ion suppression effects. Based on the past experience on ion suppression effects in both ESI (J. Mass Spectrom. 2014, 49, 639) and MALDI systems (Rapid Commun. Mass Spectrom. 2019, 33, 327), Gongyu was excited to observe signal enhancements with NBA modification of numerous biomolecules, initially focusing on neuropeptides and large proteins. Aside from signal improvement, a more intriguing observation was the detection of several new peaks generated from the mixed MALDI-MS cocktail. Further examination revealed that these additional peaks were due to specific NBA labeling of primary amine groups at the N terminus and lysine residues. Currently the group is developing more applications based on the unique feature of NBA-based nsPCR and more suitable systems to initiate nsPCR. Taken together, these features of nsPCR, coupling with different MS instrumentation platforms, would enable a broad range of biological applications that could benefit from in situ and rapid structural probing of various biomolecules from diverse set of samples, including single cells and comparative biological tissues from healthy and disease conditions. To read the full article, please click here (https://www.nature.com/articles/s41467-019-12548-0).

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Lingjun Li Research Group Reunion at the 2019 ASMS Annual Conference, in Atlanta, GA.

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Dr. Gongyu Li (first author of both Nature Communication papers highlighted here) and Professor Lingjun Li at the 2019 ASMS Postdoctoral Career Development Award ceremony at the ASMS Annual Conference, in Atlanta, GA.

Ø Figures for LRG CASMS Digital Committee:

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Figure 1. Chiral peptide chemistry involved in AD. D-isomerized Aβ could be a new target due to its distinct monomer structure, oligomerization behavior and receptor recognition feature. 

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Figure 2. The overall concept oftheiCAP. a) Metal-enhanced multidimensional epimeric discrimination to amplify the D/L structural differences (DLSDs) of Aβ monomers. b) Discrimination of chiral Aβ fragment oligomers by assembly/growth curve based multidimensional DLSD comparisons. c) Elucidation of chiral Aβ-receptor recognition by CIU fingerprint and SPR-based kinetic evaluation. d) The calculation equations for DLSD.CCS values of apo-Aβ fragment;CCS values of [Aβ + Cu(II)] complex; CCS values of [Aβ + 2Cu(II)] complex.CCS values of Aβ oligomers (aggregation number = n). 

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Figure 3. The overall concept of nanosecond photochemical reaction (nsPCR), simultaneously enabling high-throughput, highly efficient protein structural probing based on surface accessible amine labeling and on-demand matrix removal at nanosecond and micrometer environment. 

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