Biology subjects

Truong, D.-J. J.

Publications and source records attributed to Truong, D.-J. J..

3 recordsLinked to original sources

bioRxiv2025

Improving proteomic dynamic range with Multiple Accumulation Precursor Mass Spectrometry (MAP-MS)

Orbitrap (OT) -based mass spectrometer platforms are a gold standard in high-resolution mass spectrometry, where their primary disadvantage is slower-scanning speed in comparison to time-of-flight or linear ion trap mass analyzers. In this study, we utilize long OT transients to extend the precursor dynamic range by modifying the selected ion monitoring method to multiplex several precursor m/z ranges from 400 to 1000 m/z into a single scan called "Multiple Accumulation Precursor Mass Spectrometry" (MAP-MS). Our approach requires no software or hardware modifications and hides the additional ion accumulation steps during the time it takes to make other Orbitrap measurements, producing precursor spectra with nearly 2x dynamic range and essentially no consequences. We collected data using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) methods to evaluate a range of approaches. With DDA, MAP-MS precursor quantification improves with higher quality measurements. At the same time, DIA detection is enhanced by up to 11% when combining precursor and tandem mass spectra for peptide detection.

biochemistry↗

bioRxiv2025

Genetic Manipulation of Mammalian Cells in Microphysiological Hydrogels

Engineering functional 3D tissue constructs is essential for developing advanced organ-like systems, with applications ranging from fundamental biological research to drug testing. The generation of complex multicellular structures requires the integration of external geometric and mechanical cues with the ability to activate genetic programs that regulate and stimulate cellular self-organization. Here, we demonstrate that gelatin methacryloyl (GelMA) hydrogels serve as effective matrices for 3D cell culture, supporting both in situ genetic manipulation and cell growth. HEK293T cells embedded in GelMA remained viable and proliferated over 16 days, forming clusters within the matrix. We achieved efficient gene delivery in the 3D hydrogel environment using both plasmid DNA and mRNA as gene vectors. Furthermore, we applied in situ prime editing to induce permanent genetic modifications in embedded cells. To achieve spatially confined gene expression, we introduced gel-embedded channels that allowed localized stimulation via doxycycline perfusion through a Tet-On system. Our findings establish GelMA hydrogel matrices as a versatile platform for generating spheroidal cell cultures while enabling precise genetic control and spatially resolved cellular manipulation through diffusible cues.

bioengineering↗

bioRxiv2024

Non-destructive transcriptomics via vesicular export

Transcriptomics enables comprehensive, multiplexed characterization of cellular states, yet prevailing methods typically require cell fixation or lysis, precluding longitudinal analysis of RNA expression in living cells. Here, we present non-destructive transcriptomics by vesicular export (NTVE), a platform for multi-time-point monitoring of RNA expression dynamics in living cells. Stabilized RNA reporter barcodes can be selectively packaged and exported from cells via virus-like particles (VLPs) bearing bioorthogonal affinity handles for convenient multichannel tracking of co-cultured cells. Using an engineered poly(A)-binding protein adapter, NTVE exports endogenous transcripts from inducible human and murine cell lines with high concordance to conventional lysate-derived RNA-seq. NTVE captures transcriptome changes in response to genetic and chemical perturbations within the same cells over time using standard sequencing workflows. NTVE can further be equipped with fusogens to deliver mRNA-encoded effectors or ribonucleoprotein gene editors from sender cells, activating gene reporters in co-cultured recipient cells. We demonstrate the utility of NTVE for monitoring hiPSC differentiation through daily non-destructive transcriptomic profiling of lineage-specific marker dynamics.

bioengineering↗