LimsLab @ NKU

Rapid: Nanosecond Photochemical Structure Probing

Dr. Li has the long-standing research interest focusing on the development and application of ultrafast separation techniques capable of in situ purification and identification of native proteins directly from living cells. As such, one needs to in situ remove intracellular and extracellular matrices surrounding cell-trapped target proteins, where the separation time scale should be as short as possible to maintain the biologically relevant structural information. During his PhD career, Gongyu has developed a ultrafast in-solution separation method and integrated into MS (Li et al. Anal. Chem. 2016, 88, 10860-10866), and has demonstrated its utility for several protein systems directly from living cells (Li et al. Anal. Chem. 2018, 90, 3409-3415). In this manner Dr. Li has assembled both ultrafast separation and online electroporation-induced protein release into a small region inside a nanospray needle. In a recent study (Anal. Chem. 2019, 91, 10441-10447), Dr. Li and coworkers have provided further evidence of ultrafast separation. Meanwhile, Dr. Li’s ultrafast separation work facilitated the mechanistic investigation of click chemical reactions (Chem. Sci. 2017, 8, 214-222). Benefiting from Gongyu’s contribution to improved understanding of the basic mechanisms and early applications of ultrafast separation, this separation technique has recently been used for in situ chemical profiling of single living neurons (PNAS, 2018, 114, 2586-2591) and for revealing a novel glutamate biosynthetic pathway in the brain (Cell, 2018, 173, 1716-1727). In addition to contribution to the solution-phase separation improvement, Gongyu also devoted himself in the development of online gas-phase structural technique, such as the development of a new regime for ion mobility-mass spectrometry (IM-MS)-based identification of soltuion-phase conformational changes (Li et al. Anal. Chem. 2018, 90, 7997-8001). Dr. Li is ambitious to combine ultrafast solution-phase separation with gas-phase structural MS tool, IM-MS, serving as an emerging alternative to probe more structural details of native proteins directly from living cells.

While Dr. Li is much interested in serving as human disease fighter, he believes the high-quality technological and analytical developments are of most fundamental and prerequisite importance before biologist and us can take any further actions facing cutting-edge challenges. Photochemical reactions (PCRs) have long been widely involved in many aspects from fundamental research to industry-wide applications. In another paper (Li et al. Nat. Commun. 2019, 10, 4697, FEATURED by Nature Communications Editors HIGHLIGHTS), he demonstrated the suitability and possibility to adapt nanosecond PCR (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 and achieved in a few nanosecond time scale.

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, Dr. Li 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-terminus and lysine residues of proteins. The on-demand matrices 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% with a high-throughput feature. 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. Inspired by this JACS paper, Dr. Li began to explore its utility in matrix-assisted desorption ionization (MALDI)-MS in terms of alleviating ion suppression effects. Notably, Dr. Li 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), Dr. Li was excited to observe signal enhancements with the help of NBA modification for 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.

We envision many more applications will be easily established based on the unique feature of NBA-based nsPCR, and further development of more suitable systems to initiate nsPCR will greatly expand its general applicability as a rapid and high-throughput structural probe. 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.

Lossless: AIU/AIF IM-MS

MS-based composite strategies have considerably dominated gas-phase glycoprotein 3D structural analysis due to their inherent and indispensable capability in resolving glycan and glycoprotein microheterogeneity, which has become even more versatile along with the continuous developments in native MS, tandem MS including collision-induced unfolding (CIU), and ion mobility interfaces. Native ion mobility-mass spectrometry (IM-MS) has emerged as a top choice for protein structure study at the accurate molecular-mass scale, especially with the recent remarkable advancements in instrument resolving power, protein activation and manipulation techniques, and multiple data processing and visualization methods. Notably, charge state selection has been widely acknowledged as a determinant consideration to obtain reliable and biologically relevant structural information. Multiple independent research lines have reported the common observation of charge state-dependent structures and topologies of nanospray generated protein ions. The unique gas-phase charge-state-distribution phenomenon that is distinct from native solution phase, not only adds uncertainties of the structural gaps between these two phases, but also increases the time cost and degrades the confidence of gas-phase structural measurements.

To effectively and confidently dissect the PTM effects on the overall 3D arrangements of glycoproteins with high-level microheterogeneity, we introduce Native IM-based Lossless Structural MS (LSMS) strategy. To overcome the barrier of multistep, time-consuming charge-selection/optimization processes and charge-bias effects on protein structures, the LSMS strategy adopts a “charge selection-free” concept, in which all forms of protein ions’ structures and topologies are accounted for, and no precursor ion selection is mandatory. Namely, all ions are diverted into the activation cell. In this way, we manage to perform all ion unfolding/fragmentation (AIU/AIF) sequentially in a charge-independent manner on sialylated glycoproteins. The LSMS additionally relies on the further modifications of data visualization and quantitative characterization based on the CIUSuite and UniDec. By using a model sialylated glycoprotein transferrin (TF), LSMS allows for not only the reliable tracking of rapid, efficient and in situ releasing of sialic acids and the simultaneous, charge-independent stepwise unfolding of native-like glycoproteins, but also unprecedentedly revealing the differential roles of Neu5Ac and Neu5Gc sialylations in fine-tuning of glycoprotein structures and topologies.

These results not only provide new molecular signatures for the preclinical functions of PTMs, but also indicate that inhibitors that sustain Neu5Ac level at suitable values could be pursued as potential therapeutics to slow the progression of several sialylated glycoprotein-linked diseases, including AD. Furthermore, we envision that the preclinical implications and underexplored molecular basis of glycosylation other than terminal sialylations attainable using our LSMS platform will find promising applications in other target glycoproteins in both health and disease conditions. It will be of great interest to apply the current strategy to revealing molecular and structural basis for the association of the O-GlcNAcylation of such glycoproteins with AD progression, as well as the linkage of the enhanced secretion of amyloid β peptides to abnormal sialylation of amyloid β-precursor protein

High-resolution: Structural Amplification MS

Of concern is the recent failure of several clinical trials on AD, highlighting the urgent need for earlier, possibly preventative intervention, and raising the question of what form of Aβ is the best target (Prion 2014, 8, 119). Chiral inversion of amino acids is thought to modulate the structure and function of many neuropeptides (Science 1987, 238, 200/Biochem. Biophys. Res. Commun. 1997, 240, 354/ Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 413/Bioanal. Rev. 2009, 1, 7/), 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 (J. Chromatogr. B 2011, 879, 3141/J. Neuropathol. Exp. Neurol. 2003, 62, 248), there is a possibility that D-isomerized Aβ play a vital role in AD pathogenesis and development. However, since Aβ D-isomerization is slowly progressed, age-dependent and is present at low stoichiometry (e.g. less than 10%, Neuroscience 2001, 104, 1003), the role of chiral Aβ has long been ignored and largely underexplored, in part due to lack of effective tools. Recent studies (Nature 2018, 554, 249/J. Am. Soc. Mass Spectrom. 2019, 30, 1325) further highlighted the long-term ignoration, and the importance, of stereoisomeric Aβ isoforms, through pinpointing the isoform-dependent antibody selection during Aβ isolation and sampling from AD brains. Towards this end, Dr. Gongyu Li present an innovative conception and potentially new drug target for Alzheimer’s disease (AD) therapy by investigating chiral effects on Aβ peptide. In the paper published in Nature Communications (Nat. Commun. 2019, 10, 5038), Dr. Li developed an integrated, multidimensional ion mobility-mass spectrometry (IM-MS)-based approach to study chirality-regulated structural features of Aβ fragments and their influence on Aβ receptor recognition.

Dr. Li developed and established an innovative analytical platform, benefiting from the rational designs that target Aβ chiral chemistry (Figure 1). 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 crosstalking 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, while there are some previous efforts targeting to key interaction sites of wild type Aβ through the design of D-amino acid peptide inhibitors (Prion 2014, 8, 119), our study just for the first time highlighted the importance of considering key amino acid chirality in Aβ, when designing any types of inhibitors targeting to disrupting or directing 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, in terms of many aspects 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 his own research experience on zinc finger peptide-zinc binding (J. Am. Soc. Mass Spectrom. 2017, 28, 2658) and other IM-MS-based peptide-metal binding studies (J. Am. Soc. Mass Spectrom. 2017, 28, 1293). These previous studies suggested that metal binding is dependent on the structural features of peptide itself and can enhance structural difference amongst peptide isomers. Dr. Li therefore selected copper as first candidate metal, as it has its unique isotopic distribution that helps us identify the binding events much easier even only with MS1 measurements (e.g. no MS/MS is mandatory). Besides, Cu2+ binds to most peptides with a moderate to high affinity and thus we can capture such types of binding in the gas phase even after desolvation. Lastly, it is also widely involved in many real biological situations which may link our experiments biologically to some real disease or human health case. The next question, however, comes to how to maximize the chiral amplification power and how to quantitatively characterize/report such chiral amplification. To this end, Dr. Li developed a multidimensional data visualization method with rational choice of individual coordinates/vectors, namely, the collisional cross-sections (CCSs) 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. Furthermore, Dr. Li decided to use the spatial distance in this 3D scattering space to quantitatively characterize the D/L Structural Difference (DLSD). The DLSD method has now been proven very useful in this type of evaluations. We also adopted the same conception for Aβ oligomer chiral amplification and quantitative characterization. The authors named the whole set of this innovative IM-MS-based analytical strategy as integrative chirality anatomy platform (iCAP, Figure 2).

Looking into the near future, Dr. Li plans to extend the fragment-based in vitro study to full-length Aβ in vivo exploration, starting with some in cell tests. Notably, we anticipate combining this iCAP conception with our previous D-amino acid residue localization method (Anal. Chem. 2014, 86, 2972). This combined follow-up study can contribute to a systematic interrogation of chiral inversion of various amino acid residues on full-length Aβ stereoisomers and allowing precise localization of the D-amino acid residues. Ultimately, we hope to provide more concrete molecular basis for potentially new AD drug targets, including the previously ignored structural differences caused by amino acid chirality. A bold idea is, if we found D-isomerized Aβ with significantly decreased cytotoxicity, directed chiral therapy on Alzheimer’s disease patients through precise control of site-specific (e.g. Ser- and Asp-residues) Aβ D-isomerization enzymes.

Future SMS: Targeted Proteomics-guided Native SMS

Next-generation Native SMS should smartly and comprehensively integrate the individual benefits and advantages of ambient MS (in situ MS), traditional native IM-MS and targeted proteomics. We are dedicated to pushing native IM-MS more and more "NATIVE", with special focus on technical improvements on analytical speed, sensitivity and throughput. These fundamental MS research should be indispensable prerequisite of reporting more "real" and closer to biological circumstances, thus facilitating MS to be developed as something really meaningful to human disease fighting through protein-level drug-linked and receptor-related fundamental research and industrial applications.