Huilin Li

As the American Chemical Society marks its 150th anniversary, it is a fitting moment to reflect on how a young analytical discipline, proteomics, matured into a central pillar of the chemical and biological sciences and how the Journal of Proteome Research (JPR) chronicled and guided that transformation. Founded at the dawn of shotgun proteomics, JPR has published pivotal advances in separation, ionization, computational interpretation, quantitative strategies, and biological applications. In this Perspective, we trace that arc from the first near-complete eukaryotic proteomes through the quantitative and post-translational era, the computational turn, and the current high-throughput, integrative, and artificial-intelligence-assisted phase. Along the way, we highlight how the same infrastructure enabled mass spectrometry-based metabolomics, yielding an integrated framework for molecular phenotyping.

The rapid evolution of SARS-CoV-2 necessitates a deeper understanding of antibody neutralization mechanisms to guide the development of broad-spectrum therapies. Here, we integrate pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS), biolayer interferometry (BLI), and negative-stain electron microscopy (nsEM) to dissect the dynamic regulation of two engineered spike (S) protein constructs, the prefusion-stabilized S-6P and the wild-type-like S-R, by two antibody classes: the noncompetitive RBD class V antibody R1-32 and the competitive class I antibody B38. Our results show that both spike's native receptor, human angiotensin-converting enzyme 2 (ACE2), and B38 primarily protect epitopes within the receptor-binding domain (RBD), inducing a similar level of fusion-priming structural transitions without causing trimer disassembly. In contrast, R1-32 binds to a semicryptic epitope and uniquely triggers long-range allosteric destabilization across the "tripartite interface", a conserved structural hub centered around Domain C. This local perturbation propagates through region 962-977 to the trimer interface, driving time-dependent S-trimer disassembly, as further supported by nsEM. Combining these insights, eight fully conserved residues within the tripartite interface were identified as critical hotspots, representing potential targets for broad-spectrum antibodies capable of inducing trimer disassembly. Together, these findings establish a structure-dynamics framework for rational antibody design, emphasizing the targeting of conserved, conformation-sensitive epitopes to outpace viral immune evasion. Moreover, this work highlights pulsed HDX-MS as a powerful approach for resolving antibody-mediated dynamic regulation of the SARS-CoV-2 spike protein, facilitating the development of next-generation broad-spectrum therapeutics against emerging variants.

Histone deacetylase 8 (HDAC8) is a well-known epigenetic regulator for cancer therapy. However, developing targeted inhibitors for HDAC8 is challenging due to a limited understanding of its structural dynamics, which is crucial for ligand interaction. Here, we employed an integrated approach, including native mass spectrometry (native MS), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics (MD) simulation, to investigate the inhibition mechanism and dynamic regulation of human HDAC8 (hHDAC8) by selective inhibitor PCI-34051, compared with the pan-inhibitor SAHA. Our results revealed that PCI-34051 engages with an expanded set of residues and conforms more aptly to the binding channel of hHDAC8, stabilizing the flexible loops surrounding the binding channel. Moreover, this dynamic stabilization effect is not limited to the binding regions, but also extends to distant regions (such as L2, α5, and α1 + α2), with L3 serving as a critical structural bridge. Overall, these results show the structural and dynamic regulations of hHDAC8 by PCI-34051, which induces a lower energy state for the protein-ligand system compared to SAHA, thus showing better inhibitory effects. In addition, it also suggests that certain regions, specifically loops L2 and L3, within the hHDAC8 protein could be key regions for targeted intervention.

Cross-linking mass spectrometry (XL-MS) is widely used in the analysis of protein structure and protein-protein interactions (PPIs). Throughout the entire workflow, the utilization of cross-linkers and the interpretation of cross-linking data are the core steps. Cisplatin, as a well-known anticancer drug, has been previously demonstrated for its capability and advantages as a cross-linker. However, the complexity of platinum(II) cross-linked products and the lack of suitable software for data interpretation have hindered its further application. In this work, a two-stage data analysis strategy for platinum(II) cross-linked peptides has been developed and demonstrated on a pair of phosphorylation-induced allosteric systems, glycogen phosphorylase (GP) b and a. This two-stage data analysis strategy takes into account the identification of various types of Pt(II)-containing fragment ions and incorporates the unique isotope distribution properties of Pt(II)-based cross-linkers to eliminate false-positive data and achieve accurate identification of Pt(II)-based cross-linked peptides. The Pt(II)-based cross-linking results allow the capture of structural differences between GPb and GPa at the N-terminus and the tower-tower helices interface, which is consistent with the X-ray crystallography structure as well as our previous HDX-MS results. In addition, it also complements the structure of noncrystallizable regions. Finally, through discussion of existing data search engines and issues in spectral analysis of Pt(II)-based cross-linked peptides, we put forward proposals for follow-up software design, cross-linker developments, and guidance for the application of platinum(II)-based drugs. Overall, Pt(II)-based XL-MS can be a useful tool to complement both experimental and computational structural biology.

Cross-linking mass spectrometry (XL-MS) is widely used in the analysis of protein structure and protein-protein interactions (PPIs). Throughout the entire workflow, the utilization of cross-linkers and the interpretation of cross-linking data are the core steps. In recent years, the development of cross-linkers and analytical software mostly follow up on the classical models of non-cleavable cross-linkers such as BS3/DSS and MS-cleavable cross-linkers such as DSSO. Although such a paradigm promotes the maturity and robustness of the XL-MS field, it confines the innovation and flexibility of new cross-linkers and analytical software. This critical insight will discuss the classification, advantages, and disadvantages of existing data analysis search engines. Take the new platinum-based metal cross-linker as an example, potential pitfalls in characterization of cross-linked peptides using existing software are discussed. Finally, ideas on developing more flexible, comprehensive, and user-friendly cross-linkers and software tools are proposed.

Allosteric regulation plays a fundamental role in innumerable biological processes. Understanding its dynamic mechanism and impact at the molecular level is of great importance in disease diagnosis and drug discovery. Glycogen phosphorylase (GP) is a phosphoprotein responding to allosteric regulation and has significant biological importance to glycogen metabolism. Although the atomic structures of GP have been previously solved, the conformational dynamics of GP related to allostery regulation remain largely elusive due to its macromolecular size (∼196 kDa). Here, we integrated native top-down mass spectrometry (nTD-MS), hydrogen-deuterium exchange MS (HDX-MS), protection factor (PF) analysis, molecular dynamics (MD) simulations, and allostery signaling analysis to examine the structural basis and dynamics for the allosteric regulation of GP by phosphorylation. nTD-MS reveals differences in structural stability as well as oligomeric state between the unphosphorylated (GPb) and phosphorylated (GPa) forms. HDX-MS, PF analysis, and MD simulations further pinpoint the structural differences between GPb and GPa involving the binding interfaces (the N-terminal and tower-tower helices), catalytic site, and PLP-binding region. More importantly, it also allowed us to complete the missing link of the long-range communication process from the N-terminal tail to the catalytic site caused by phosphorylation. This integrative MS and in silico-based platform is highly complementary to biophysical approaches and yields valuable insights into protein structures and dynamic regulation.