Yi-Kai Liu

Light is an important environmental stimulus that is necessary for vision and circadian entrainment. Here, we present a system-wide analysis of the kinase-mediated light response in Drosophila using optimized data-independent acquisition mass spectrometry (DIA-MS) for quantitative phosphoproteomics analysis. Exposure to standard white light or high-intensity blue light induced a widespread increase in phosphorylation relative to dark-adapted flies. The light-sensitive phosphoproteins were part of multiple pathways, including phototransduction, circadian control, lipid metabolism, and synapse organization, and exhibited distinct phosphorylation signatures under dark adaptation or white or blue light. The Ca ²⁺ -permeable transient receptor potential (Trp) channel was differentially phosphorylated at multiple sites in a white and blue light–dependent manner, and phosphorylation stoichiometric analysis identified Thr ⁸⁴⁹ in Trp as a pivotal functional switch. It was entirely unphosphorylated in the dark, but 80% of the Trp molecules were phosphorylated in either light condition. This phosphorylation event was mediated in part by the eye-specific protein kinase C InaC and regulated the eye’s electrophysiological response to oscillating light. Thus, our results show that light remodels the Drosophila phosphoproteome, establishing a crucial mechanism for sensory adaptation to a changing light-dark environment.

Extracellular vesicles (EVs) have gained increasing attention with their intriguing biological functions and their molecular cargoes serving as potential biomarkers for various diseases, including cancers. A relatively lower abundance of EV proteins compared to cellular counterparts necessitates sensitive and accurate quantitative proteomic strategies. Multiplexed proteomics combined with data-independent acquisition (mDIA) has shown promise for improving sensitivity and quantification over traditional DDA and label-free methods. Despite this, mDIA pipelines that utilize various types of spectral libraries and search software suites have not been thoroughly evaluated with EV proteome samples. In this study, we aim to establish a robust mDIA pipeline based on dimethyl labeling for quantitative proteomics of EVs. EVs were isolated using the extracellular vesicle total recovery and purification (EVtrap) technique and processed directly through an on-bead one-pot sample preparation workflow to obtain digested peptides. We evaluated different mDIA pipelines, including library-free and library-based DIA on the timsTOF HT platform. Results showed that library-based DIA, with project-specific spectral libraries generated from StageTip-based fractionation, outperformed other pipelines in protein identification and quantification. We demonstrated for the first time EV proteome landscape changes caused by the IDH1 mutation and inhibitor treatment in intrahepatic cholangiocarcinoma, highlighting the utility of mDIA in EV-based biomarker discovery.

Plants are constantly exposed to volatile organic compounds (VOCs) that are released during plant-plant communication, within-plant self-signaling, and plant-microbe interactions. Therefore, understanding VOC perception and downstream signaling is vital for unraveling the mechanisms behind information exchange in plants, which remain largely unexplored. Using the hormone-like function of volatile terpenoids in reproductive organ development as a system with a visual marker for communication, we demonstrate that a petunia karrikin-insensitive receptor, PhKAI2ia, stereospecifically perceives the (−)-germacrene D signal, triggering a KAI2-mediated signaling cascade and affecting plant fitness. This study uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).

The ability to monitor the response of metabolic enzymes to drug exposure in individuals is highly appealing and critical to personalized medicine. Although pharmacogenomics assesses genotypic differences, it does not report changes in metabolic enzyme activities due to environmental factors such as drug interactions. Here, we report a quantitative proteomics strategy to monitor drug metabolic pathways by profiling metabolic enzymes in circulating extracellular vesicles (EVs) upon drug exposure. Mass spectrometry (MS)-based measurement revealed that changes in metabolic enzyme abundance in EVs paralleled those in hepatic cells isolated from liver tissue. Coupling with multiplexed isotopic labeling, we temporally quantified 34 proteins involved in drug absorption, distribution, metabolism, and excretion (ADME) pathways. Out of 44 known ADME proteins in plasma EVs, previously annotated mouse cytochrome P450 3A11 (Cyp3a11), homolog to human CYP3A4, and uridine 5'-diphospho (UDP) glucuronosyltransferase 2A3 (Ugt2a3), increased upon daily rifampicin dosage. Dasatinib, a tyrosine kinase inhibitor to treat leukemia, also elevated Cyp3a11 levels in plasma EVs, but to a lesser extent. Altogether, this study demonstrates that measuring drug enzymes in circulating EVs as an effective surrogate is highly feasible and may transform today's drug discovery and development for personalized medicine.