Keskitalo, S. E. | bioRxiv Science

bioRxiv2025

ISG15 Differentially Modulates Clade Ib and II MPXV Infection in MEF cells

The unprecedented human-to-human transmission of Clade IIb monkeypox virus (MPXV) during the 2022 outbreak has renewed focus on host determinants of viral fitness. Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein with broad immunomodulatory functions, yet its role in MPXV infection remains unclear. Using representative strains from recent and historical outbreaks spanning Clades I and II, we show that ISG15 deficiency enhances viral replication and protein production in murine cells. Given that rodents are considered potential natural reservoirs of MPXV, these findings highlight the importance of studying murine models to understand virus-host interactions. Notably, the 2024 Democratic Republic of Congo strain displays reduced sensitivity to ISG15, suggesting clade-specific adaptation. ISG15 also influences viral immune evasion, as knockout cells infected with Clade II viruses expressed fewer immunomodulatory proteins and exhibited marked reductions in host protein phosphorylation. These results identify ISG15 as a determinant of MPXV infection and underscore evolutionary differences between clades.

microbiology↗

bioRxiv2025

Automated MagNet Enrichment Unlocks Deep and Cost-Effective LC-MS Plasma Proteomics

Plasma is an ideal material for proteomics due to its diverse protein content reflecting physiological and pathological states, and its compatibility with minimally invasive sampling. Deep proteomic profiling of plasma is hindered by the dominance of high-abundant proteins that mask the detection of low-abundant proteins. To overcome this, we compared five plasma protein enrichment methods, MagNet, ENRICHplus, ENRICHiST, EasySep, and EXONET, against neat plasma using LC-MS proteomics. All five methods substantially increased protein identifications, with MagNet, ENRICHplus, EasySep, and EXONET yielding up to 4200 proteins per sample, over 7-fold more than neat plasma, using a 44-minute gradient on the Evosep One and data-independent acquisition on the timsTOF Pro 2. These methods enriched extracellular vesicle-associated proteins while effectively depleting high-abundant proteins. We further optimized the cost-effective MagNet protocol by increasing the plasma-to-bead ratio and automated the workflow, including Evotip loading, on the Biomek i5 liquid handler. Automated MagNet demonstrated high reproducibility and a remarkably low total cost of just a few dollars per sample. This streamlined enrichment strategy enables scalable, high-throughput LC-MS plasma proteomics, supporting biomarker discovery across large clinical cohorts.

systems biology↗