Meng-Ju Wu

Quantitative proteomics requires robust, accurate labeling strategies that minimize sample handling and preserve analytical sensitivity. Enzymatic 18O labeling offers a simple, universally applicable alternative to other isotopic labeling methods, but has been limited by incomplete labeling, back-exchange, and reliance on post-digestion, in-solution 18O labeling workflows that are impractical and introduce cumulative sample losses. Here, we present a fully integrated platform combining single-pot solid-phase-enhanced sample preparation (SP3) with direct on-bead trypsin-catalyzed C-terminal 18O labeling (SP-18O) during proteolytic digestion and data-independent acquisition mass spectrometry (DIA-MS). By performing 18O incorporation directly on-bead during digestion, the SP-18O approach eliminates the need for a separate post-digestion labeling step and associated sample losses compared to conventional enzymatic labeling workflows. Integration with DIA-MS further improves identification confidence and quantitative performance through distinctive y-ions. The platform is validated on two biologically distinct sample types, extracellular vesicles (EVs) secreted from model cancer cells and immunoaffinity-enriched protein complexes from Drosophila head tissue, demonstrating robustness and generalizability. This integrated strategy offers a practical, cost-effective solution for accurate quantitative proteomics while minimizing sample loss and analysis time.

List of contents of each Supplementary Table

Supplementary Table S1. List of SsTOP genes Supplementary Table S2. List of SsTOP (-E2F) genes Supplementary Table S3A. TF activity DMSO vs PKAi Supplementary Table S3B. TF activity PKAi+SIKi (COMBO) vs PKAi Supplementary Table S3A. CRTC2 targets gene set Supplementary Table S4. Curated FLC gene sets Supplementary Table S5. Expression of amino acid metabolism/urea cycle genes in FLC Supplementary Table S6. Regulation of curated FLC pathways by PKAi and SIKi in FLX1 Supplementary Table S7. MSigDB (Human) Hallark_E2F targets gene set

Supplementary Figure S1: Validation of the specificity of the p-SIK Ser343 antibody. Supplementary Figure S2. PKAfus inactivates SIKs to support tumor cell growth in vitro and in vivo. Supplementary Figure S3. PKAfus reprograms transcription by inactivating SIK and stimulating CRTC2. Supplementary Figure S4. CRTC2/CREB mediates transcriptional regulation downstream of PKAfu. Supplementary Figure S5. CRTC2/CREB supports tumor cell growth downstream of PKAfus. Supplementary Figure S6. Primary FLC tumors show deregulation of SIK/CRTC2- controlled genes. Supplementary Figure S7. PKAfus driven tumors are sensitive to p300 inhibition. Supplementary Figure S8. PKAfus driven tumors are sensitive to p300 inhibition.

Tumor cells respond and adapt to environmental stresses that facilitate growth in hostile environments, including cytokine-mediated inflammation elicited by antitumor immunity and enhanced by immune checkpoint blockade (ICB). However, cytokine responses also induce transcriptional and cell-state changes that may predispose tumor cells to new vulnerabilities, which remain poorly explored. Here we performed in vitro genome-scale CRISPR loss-of-function screens in eight cancer models exposed to interferon-γ (IFNγ), interferon-β or tumor necrosis factor to map inflammation-induced genetic vulnerabilities. We identified members of the glycosylphosphatidylinositol (GPI) transamidase complex and the lipid phosphatase FITM2 as interferon-specific cancer dependencies. Tumor-specific deletion of GPI transamidase subunits or FITM2 markedly enhanced response to ICB in vivo. By integrating functional genomics, metabolomics and pharmacologic perturbation of downstream stress pathways, we found that loss of FITM2 predisposed cancer cells to IFNγ-driven endoplasmic reticulum and oxidative stress, culminating in paraptosis-like cell death. Collectively, these findings identify tumor-intrinsic dependencies governing responses to inflammatory cytokines.

Mutations in isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are common in multiple types of human cancer and cause accumulation of the oncometabolite D -2-hydroxyglutarate (D2HG) instead of α-ketoglutarate, driving cancers like gliomas and acute myeloid leukaemia by blocking cell differentiation and promoting tumour growth. Here we discovered protein O -2-hydroxyglutarylation by D2HG using chemical proteomics and further revealed distinct chiral preferences for D2HG and L -2-hydroxyglutarate (L2HG) modifications. D2HG modifications are upregulated in IDH-mutant cells or upon D2HG treatment, while L2HG modifications increase under hypoxic conditions or following L2HG treatment. Notably, two kinases MRCKA and SLK are modified by D2HG and L2HG, respectively, and confirmed by synthetic peptide standards. Phosphoproteomics revealed reduced phosphorylation of MRCKA and SLK substrates, suggesting crosstalk between D/L-2HG modification and kinase activity. These findings highlight distinctive roles of D/L-2HG modifications in cancer progression and suggest potential avenues for therapeutic targeting of oncometabolite-induced post-translational modifications.

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