Contact

  • Academic
    mgreeson@stanford.edu
    University - Student Department: Department of Molecular and Cellular Physiology Position: Graduate

All Publications

  • Dissociation kinetics and avidity gate SARS-CoV-2 neutralization by HR2 stem helix antibodies. bioRxiv : the preprint server for biology Crivelli, V., Guerra, C., Abernathy, M. E., Sgrignani, J., Zoppi, G., Greeson, M. L., Sanga, A., Locatelli, P., Cantergiani, J., Cena, B., Rincón, T. C., Lee, Y. E., Eso, M., Jarrossay, D., Biggiogero, M., Calvaruso, V., Pellanda, A. F., Garzoni, C., Tamagnini, E., Lestani, S., Varani, L., Sommer, S., Fernandez, D., Barba-Spaeth, G., Niejadlik, E. G., Bournazos, S., Robbiani, D. F., Barnes, C. O., Cavalli, A. 2026

    Abstract

    SARS-CoV-2 evolution has reduced the efficacy of clinical monoclonal antibodies, underscoring the need for therapeutics targeting conserved viral regions. The Spike (S) heptad repeat 2 (HR2) stem helix is highly conserved across SARS-CoV-2 variants and related betacoronaviruses. Although neutralizing antibodies to this region have been identified, the evolution of humoral responses to HR2 and the determinants of effective neutralization at this site remain poorly understood. We previously identified human neutralizing antibodies to a conserved peptide within this region (HR2 coldspot). Here, longitudinal analysis over 30 months shows that HR2-specific antibodies persist and undergo somatic hypermutation, yet antibodies isolated at later time points did not surpass the breadth or potency of hr2.016, which emerged shortly after primary infection. Crystal structures of four HR2 stem-helix antibodies revealed convergent recognition across distinct antibody lineages. However, comparison of hr2.016 with its non-neutralizing clonal relative hr2.086 showed that adopting this shared binding mode is not sufficient for effective neutralization. Characterization of this antibody pair through surface plasmon resonance and molecular dynamics simulations revealed that robust neutralization requires slow intrinsic dissociation reinforced by avidity. Together, these findings show that continued evolution of HR2-specific responses does not necessarily enhance antibody breadth or potency and that, despite convergent epitope recognition, effective neutralization requires slow intrinsic dissociation reinforced by avidity, highlighting kinetic stability as a key criterion for antibody discovery and vaccine design against viral epitopes.

    View details for DOI 10.64898/2026.08.17.745160

    View details for PubMedID 42818197

    View details for PubMedCentralID PMC13622405

  • Systematic optimization and benchmarking of synchro-PASEF for high-throughput phosphoproteome profiling. bioRxiv : the preprint server for biology Brademan, D. R., Mullarkey, A. A., Greeson, M. L., Szvetecz, S., Vitek, O., Blythe, E. E., Huttenhain, R. 2026

    Abstract

    High-throughput data-independent acquisition (DIA) workflows paired with short chromatographic separations are increasingly adopted for systems biology and clinical proteomics. However, narrower peak widths from rapid separations demand faster mass spectrometer cycle times to maintain quantitative depth and reproducibility. The synchro-PASEF acquisition mode on timsTOF mass spectrometers diagonally scans across ion mobility and m/z space, enabling efficient sampling of the precursor ion cloud with shortened cycle times. While synchro-PASEF has demonstrated competitive identification depth for global protein abundance samples compared to conventional dia-PASEF, its performance for phosphoproteomics-where the precursor ion cloud is characteristically broader and bimodally distributed-has not been evaluated. Here, we systematically optimized synchro-PASEF methods for phosphoproteomics and benchmarked performance against two dia-PASEF methods across three sub-hour separations. We found that synchro-PASEF performance depends critically on balancing diagonal window number, total isolation width, and gradient length, with longer gradients favoring more windows for selectivity and shorter gradients favoring fewer windows to preserve sampling frequency. An optimized configuration quantified over 19,000 localized phosphosites using a 23-minute separation. Retention time summation (RTsum) with a factor of 2 increased phosphopeptide identifications by 5-20% and reduced phosphosite-level coefficients of variation by up to 30% across all dia-PASEF and synchro-PASEF methods tested. Using beta2-adrenergic receptor (B2AR) activation as a signaling model, we demonstrate that label-free DIA phosphoproteomics can be used to model phosphoproteomics dose-response relationships, showing that synchro-PASEF and dia-PASEF produce highly concordant phosphoproteomic responses, with comparable numbers of responding phosphosites, similar effect sizes, and nearly identical predicted protein kinase A (PKA) substrates downstream of the activated B2AR. While synchro-PASEF did not surpass optimized dia-PASEF in identification depth, its comparable biological performance and amenability to post-acquisition optimization through RTsum support its utility for high-throughput phosphoproteomics. This work provides a transferable framework for synchro-PASEF method optimization and demonstrates the broad utility of retention time summation for PASEF-based phosphoproteomics workflows.

    View details for DOI 10.64898/2026.06.26.734570

    View details for PubMedID 42395363

  • Exploring non-canonical GPCR interactions using unbiased proteomics Greeson, M. L., Brademan, D. R., Pu, Y., Marley, A., Li, Q., Polacco, B., von Zastrow, M., Myers, B., Huttenhain, R. ELSEVIER. 2025

    View details for DOI 10.1016/j.mcpro.2025.101321

    View details for Web of Science ID 001631961100050