Antibody-drug conjugates (ADCs) require antibodies with both high specificity and efficient internalization, features often overlooked by conventional discovery pipelines that rely on preselected antigens and recombinant proteins. Here, we describe an integrated phenotypic platform that combines target-unbiased live-cell biopanning with in situ chemical cross-linking and mass spectrometry to concurrently identify internalizing antibodies and their membrane-bound cognate antigens in a native cellular context. Using this approach, we identified 2E7, an antibody with rapid internalization and specificity for the integrin α3β1 (ITGA3B1) heterodimer. Integrated transcriptomic and proteomic analyses revealed pronounced overexpression of ITGA3B1 across multiple solid tumors, with particularly elevated levels in aggressive bladder cancer subtypes. A 2E7-MMAE (monomethyl auristatin E) ADC exhibited potent, dose-dependent antitumor activity in bladder cancer xenograft models, leading to tumor regression and prolonging survival. This study establishes a generalizable framework for function-first ADC discovery and nominates ITGA3B1 as a promising therapeutic target in bladder cancer.

Autism Spectrum Disorder (ASD) is a biologically heterogeneous neurodevelopmental condition, presenting a major barrier to the identification of robust and translatable molecular biomarkers. Here, we employ a cross-species proteomic framework to identify conserved protein signatures associated with ASD. Quantitative proteomic profiling of brain and serum from CNTNAP2 knockout mice, integrated with serum proteomes from individuals with ASD, revealed 132 proteins consistently dysregulated across species. Functional pathway analyses implicated coordinated alterations in lipid metabolism, synaptic signaling, and immune regulation. To prioritize diagnostically informative candidates, we applied machine learning-based feature selection and identified a minimal panel of ten proteins (COL1A1, ITIH4, CLU, NID1, C5, MASP1, PON1, PLTP, HSPA5, and FETUB) that robustly discriminated ASD from control samples. Gene ontology and KEGG pathway analyses highlighted enrichment of immune regulatory pathways, synaptic transmission, oxidative stress responses, and lipid metabolic processes, consistent with emerging models linking neuroimmune dysregulation and metabolic imbalance to ASD pathophysiology. An XGBClassifier trained on this biomarker panel achieved strong performance in independent test sets (AUC = 0.75). Together, these findings establish cross-species proteomic integration combined with machine learning as a powerful strategy for uncovering conserved, biologically grounded biomarkers in ASD, providing a framework for future validation and translational development.

OBJECTIVE:The immunopeptidome, the collection of peptides bound to human leukocyte antigens (HLAs), plays a key role in initiating immune responses. HLA-B∗51:01 is an allele associated with Behçet's disease (BD). However, the role of the immunopeptidome bound to HLA-B∗51:01 in the pathogenesis of BD remains unclear. METHODS:We profiled the HLA-bound immunopeptidome using plasma samples from HLA-B∗51:01-positive BD and healthy controls (HCs). HLA-class I molecules were immunoprecipitated, and liquid chromatography-tandem mass spectrometry was performed to compare HLA-bound peptides between HLA-B∗51:01-positive BD with HCs. Potential HLA-B∗51:01-bound peptides, T cell epitopes, were then selected by the binding prediction software NetMHCpan. The immunogenicity of the selected peptides was investigated through enzyme-linked immunospot, flow cytometry, and dextramer staining. RESULTS:We analyzed the immunopeptidome established from BD using two different methods. First, BD-specific peptides were identified. Among 8008 peptides, 2306 were found only in BD patients. The BD-specific immunopeptidome preferred hydrophobic amino acids at position 2. Exome sequencing confirmed the presence of the identified representative peptides. These peptides, when presented on monocyte-derived dendritic cells from HLA-B∗51:01-positive BD patients, effectively activated T cells, causing them to secrete proinflammatory cytokines and express the degranulation marker CD107a. Additionally, BD-predominant peptides were identified, whose amount was increased in BD than in HC. BD-predominant peptides also activated T cells, leading to the secretion of proinflammatory cytokines. CONCLUSION:The immunopeptidome presented on HLA-B∗51:01-positive BD is distinct from that of HLA-B∗51:01-positive HCs and can activate T cells and secrete proinflammatory cytokines; this may contribute to the pathogenesis of BD.

X-ray repair cross-complementing protein 4 (XRCC4), a non-homologous end-joining protein involved in DNA double-strand break repair, is highly expressed in human cancer cells and tissues. A prior OGT interactome study identified XRCC4 as a candidate for O-GlcNAcylation. O-GlcNAcylation levels, a post-translational modification found on nuclear and cytosolic proteins, are also elevated in various cancers. However, the direct regulatory mechanism linking O-GlcNAcylation to XRCC4 function in cancer cells remains unclear. Here, we found that XRCC4 is O-GlcNAcylated at threonine 308, enhancing its stability by inhibiting TRIM21-mediated ubiquitin-dependent proteasomal degradation. O-GlcNAcylation elevated XRCC4 protein levels during DNA double-strand break damage, thereby conferring resistance to such damage. Additionally, XRCC4 Thr308 O-GlcNAcylation promotes cancer proliferation, invasion, and in vivo tumor growth. These findings suggest that downregulating O-GlcNAcylation on XRCC4 could be a potential therapeutic strategy to increase cancer sensitivity to chemotherapy or radiotherapy.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality, necessitating the development of novel therapeutic strategies. In the present study, we identified TP53-regulating kinase (TP53RK) as a critical regulator of CRC cell survival and proliferation using a custom clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 library screen targeting serine/threonine kinase-related genes. TP53RK was significantly overexpressed in CRC tissues and was correlated with copy number amplification. Functional validation revealed that TP53RK depletion induced DNA replication stress, apoptosis, and cell cycle arrest, independent from p53 status. Mechanistically, TP53RK stabilized cell division cycle 7 (CDC7), a key kinase regulating DNA replication origin activation, ensuring robust minichromosome maintenance complex protein (MCM) phosphorylation and replication fork progression. Disruption of the TP53RK-CDC7 axis led to reduced MCM2 enrichment at replication origins and impaired DNA replication dynamics. Moreover, TP53RK overexpression sensitized cells to DNA replication stress (aphidicolin) and CDC7 inhibition (XL413), highlighting its potential as a therapeutic strategy. These findings establish TP53RK as a pivotal regulator of DNA replication fidelity and genomic stability, thereby providing a promising therapeutic target for CRC.

Advances in antibody (Ab) phage display technologies have enabled the development of targeted therapies with enhanced precision and clinical efficacy. Here, we report a phenotypic screening strategy combining target-unbiased live-cell biopanning with in situ chemical crosslinking and mass spectrometry to identify internalizing antibodies and their cognate antigens—key features for effective antibody-drug conjugate (ADC) development. Using this approach, we identified the 2E7 antibody, which exhibits rapid internalization and high specificity for the integrin α3β1 (ITGA3B1) heterodimer, a complex overexpressed in multiple solid tumors, including bladder cancer. We generated an ITGA3B1-targeted ADC by conjugating monomethyl auristatin E (MMAE) to the 2E7 antibody, enabling selective delivery of cytotoxic payloads to ITGA3B1-positive cells. In preclinical bladder cancer models, this ADC demonstrated potent and dose-dependent antitumor efficacy, with significant tumor regression and improved survival. Our findings establish a framework for target discovery using live-cell phenotypic screening and position ITGA3B1 as a promising therapeutic target for ADC-based treatment of advanced bladder cancer. Teaser Live-cell biopanning uncovers ITGA3B1 as a target for ADC development in metastatic bladder cancer. ### Competing Interest Statement The authors have declared no competing interest.

Despite the efficacy of chimeric antigen receptor (CAR)-T cells in selected hematological malignancies, further improvement on CAR-T designs is still desirable. We hypothesize that modifying the CAR structure to enhance immunological synapse (IS) stabilization and CAR target-binding may be a feasible strategy. Here we show that the membrane protein, CD99, is critical for IS formation in T cells by mediating actin-microtubule interaction. CD99 deficiency abolishes IS formation and prevents effective in vivo T cell immunity. Mechanistically, CD99 interacts with microtubules and actins through the transmembrane and cytoplasmic domains, respectively, with which myosin and IQGAP1 interact. As such, incorporating the transmembrane and juxtamembrane domains of CD99 into the CAR structure enhances IS formation and improves the therapeutic efficacy of human CAR-T cells against lymphoma in immune-deficient mice. Our data thus suggest that CD99-mediated IS stabilization may help improve CAR design and efficacy.

Attention-deficit/hyperactivity disorder, or ADHD, is a neurodevelopmental disorder with poorly understood molecular mechanisms. Recent studies have proposed that gene expression involved in regulating synaptic transmission in the striatum may play a role in ADHD pathogenesis. To explore the molecular basis of ADHD, we utilized proteomic analysis using whole striatal tissues from early adult thyroid hormone-responsive protein-overexpressing (THRSP-OE) mice, which displayed defining characteristics of predominantly inattentive ADHD (ADHD-PI). We focused on the striatal brain region due to its critical role in the regulation of attention, motivation, and reward processing. Moreover, the striatum modulates dopaminergic pathways that are known to be impaired in ADHD. Our analysis revealed an innate overexpression of Snap25 protein in THRSP-OE mice, indicating possible alterations in the SNARE protein complex and potential neurotransmitter dysregulation. Furthermore, a binding affinity study showed reduced dopamine D1 receptor binding concentrations and pronounced low dopamine levels in THRSP-OE mice. Repeated seven-day injections of methylphenidate improved the low dopamine levels, reducing the EEG theta/beta ratio in this animal model. These findings suggest new markers specific to the ADHD-PI presentation and further support the role of Snap25 dysregulation and possible SNARE protein complex alterations in ADHD-PI.

UDP-GlcNAc serves as a building block for glycosaminoglycan (GAG) chains in cartilage proteoglycans and simultaneously acts as a substrate for O-GlcNAcylation. Here, we show that transporters for UDP-GlcNAc to the endoplasmic reticulum (ER) and Golgi are significantly downregulated in osteoarthritic cartilage, leading to increased cytosolic UDP-GlcNAc and O-GlcNAcylation in chondrocytes. Mechanistically, upregulated O-GlcNAcylation governs the senescence-associated secretory phenotype (SASP) by stabilizing GATA4 via O-GlcNAcylation at S406, which compromises its degradation by p62-mediated selective autophagy. Elevated O-GlcNAcylation in the superficial layer of osteoarthritic cartilage coincides with increased GATA4 levels. The topical deletion of Gata4 in this cartilage layer ameliorates post-traumatic osteoarthritis (OA) in mice while inhibiting O-GlcNAc transferase mitigates OA by decreasing GATA4 levels. Excessive glucosamine-induced O-GlcNAcylation stabilizes GATA4 in chondrocytes and exacerbates post-traumatic OA in mice. Our findings elucidate the role of UDP-GlcNAc compartmentalization in regulating secretory pathways associated with chronic joint inflammation, providing a senostatic strategy for the treatment of OA. Here, the authors show that reduced transport of UDP-GlcNAc to the endoplasmic reticulum and Golgi leads to increased cytosolic UDP-GlcNAc and O-GlcNAcylation in chondrocytes. This, in turn, stabilizes the transcription factor GATA4, promoting the senescence-associated secretory phenotype and exacerbating osteoarthritis.

Non-POU domain-containing octamer-binding protein (NONO) is a multifunctional member of the Drosophila behavior/human splicing (DBHS) protein family with DNA- and RNA-binding activity. NONO is highly expressed in various types of cancer, and excessive O-GlcNAcylation has also been implicated in tumorigenesis. Although recent studies revealed that NONO is O-GlcNAcylated and that this modification is involved in DNA damage repair, it remains unknown whether O-GlcNAcylation of NONO regulates cancer cell proliferation. Additionally, little is known about the effect of O-GlcNAcylation on other biological properties of NONO. In this study, we identify Thr440 as the primary NONO O-GlcNAcylation site and demonstrates its crucial role in the assembly of paraspeckles, an important subnuclear compartment that facilitates NONO-dependent transcriptional regulation in mammalian cells. Moreover, we found that O-GlcNAcylation of NONO is required to maintain the expression of genes related to microtubule cytoskeleton organization involved in mitosis and to suppress the expression of genes related to cellular response to type I interferon. Regarding the regulation of these genes, depletion of NONO O-GlcNAcylation at Thr440 significantly inhibited the proliferation of colon cancer cells. Collectively, our findings highlight NONO O-GlcNAcylation as a key regulator modulating paraspeckle formation and as a candidate therapeutic target in colon cancer.

Despite decades of research, the etiology of autism spectrum disorder (ASD) remains largely uncomprehended, probably due to its clinical and phenotypic heterogeneity. Animal models, particularly contactin-associated protein-like 2 (Cntnap2) knockout (KO) mice, have been instrumental in elucidating ASD-related neurobiological mechanisms, as they exhibit ASD-like phenotypes, such as impaired social interactions in sociability paradigms. This provides the possibility for identifying detectable protein-based biomarkers that may assist in ASD diagnosis. Herein, we implemented an integrated approach to analyze the plasma and prefrontal cortex (PFC) proteomes exclusively from Cntnap2 KO mice (n = 3) or patients diagnosed with ASD (n = 3), along with gene ontology (GO) functional enrichment and pathway analysis. Overlapping GO terms and pathways were identified from the proteomic subsets of Cntnap2 KO mice plasma and PFC after dividing differentially expressed proteins (DEPs) into upregulated and downregulated subsets. Overlapping GO terms and pathways were further identified following the comparison of the upregulated and downregulated DEPs found in the plasma of patients with ASD. Under these GO terms and pathways, two (2) common DEPs were identified: downregulated complement C1q subcomponent subunit B (C1QB) and upregulated galectin-3-binding protein (LGALS3BP). The upregulated expression of LGALS3BP, but not C1QB downregulation, in the PFC and blood of Cntnap2 KO mice (n = 12-13) were validated through Western blotting. While future investigations will include other preclinical ASD models and clinically heterogeneous human populations, overall, these preliminary findings suggest a potential role for LGALS3BP as a biomarker for ASD and support the involvement of both central and peripheral mechanisms in its pathophysiology.

B7-H3, an immune checkpoint molecule, is prominently overexpressed in various solid tumors, correlating with poor clinical outcomes. Despite its critical role in promoting tumorigenesis, metastasis, and immune evasion, the regulatory mechanisms governing B7-H3 expression, particularly in cancer stem cells (CSCs), remain elusive. In this comprehensive study, we focused on breast CSCs to uncover the transcriptional regulators driving B7-H3 overexpression. Utilizing DNA affinity purification-mass spectrometry (DAP-MS) to analyze B7-H3 promoter regions, we identified a novel set of transcription factors, including DDB1, XRCC5, PARP1, RPA1, and RPA3, as key modulators of B7-H3 expression. Functional assays revealed that targeting DDB1 with nitazoxanide significantly downregulated B7-H3 expression, subsequently impairing tumor sphere formation and cell migration in breast CSCs. These findings not only elucidate the complex transcriptional network controlling B7-H3 expression but also open new avenues for developing targeted immunotherapies aimed at disrupting CSC-driven cancer progression.

Abstract Background: We identify TP53 regulating kinase (TP53RK), which showed the highest cell growth inhibition efficiency in six colon cancer cell lines. TP53RK is overexpressed in various cancer types, including multiple myeloma and skin cancer, but the mechanism for its tumorigenesis in colon cancer is unknown. Therefore, this study aims to reveal the tumorigenesis mechanism of TP53RK, which is overexpressed in Colorectal Cancer (CRC), through global and phospho-proteomics. Method: A total 5 CRC cell lines (HT29, SW480, HCT116, H508 and CaCO2), colon normal fibroblast CRL1459, HCT116 p53 null cell and patient-derived normal organoid were used. Immunoblotting, MTT assay, Colony formation assay, Annexin-V assay and cell cycle analysis were performed for evaluation of TP53RK loss-of-function. With kinase-substrate enrichment analysis with phospho-proteome analysis, MCM2 was found to be a substrate for the kinase regulated by TP53RK. Results: High-throughput genetic screening approach have been widely applied to the study the gene function and molecular mechanism associated with tumorigenesis. By using genome-wide CRISPR/Cas9 library screening, we identify TP53RK, the first committed negative-selected gene, as a tyrosine kinase in the colorectal cancer(CRC) specific biomarker. Through proteome analysis, we found that depletion of TP53RK can hypo-phosphorylate the DNA helicase complex, MCM2 (minichromosome maintenance protein2). According to previous studies, the phosphorylation of MCM2 is regulated by CDC7 (cell division cycle7), and the abnormal states of CDC7 and MCM2 cause DNA replication disorder. Our results show that TP53RK expression is upregulated in CRC and its depletion results in decreased CDC7 expression and hypo-phosphorylation of the MCM complex. Decreased CDC7 expression due to its instability after TP53RK depletion can lead to DNA replication errors. DNA replication errors can lead cancer cells to apoptosis, suggesting that they may be a strategy for cancer treatment. In summary, we provide TP53RK as a novel prognostic and therapeutic target in CRC. Conclusion: To recapitulate briefly, our findings suggest that the interaction of TP53RK with CDC7 can regulate CDC7 protein stability and stabilized CDC7 can phosphorylate MCM2 to initiate DNA replication. In other words, because overexpression of TP53RK acts as an oncogene that promotes tumor progression in CRC, depletion of TP53RK can degrade CDC7 and induce colon cancer cells to death or regression. Citation Format: Younghee Choi, Ji-won Park, Jun-kyu Kang, Eun-ju Kim, Sang-Hyun Song, Eugene C. Yi, Tae-You Kim. TP53RK regulates DNA replication by CDC7-mediated MCM helicase phosphorylation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6988.

Although proteomics is extensively used in immune research, there is currently no publicly accessible spectral assay library for the comprehensive proteome of immune cells. This study generated spectral assay libraries for five human immune cell lines and four primary immune cells: CD4 T, CD8 T, natural killer (NK) cells, and B cells. This was achieved by utilizing data-dependent acquisition (DDA) and employing fractionated samples from over 100 µg of proteins, which was applied to acquire the highest-quality MS/MS spectral data. In addition, Data-indedendent acquisition (DIA) was used to obtain sufficient data points for analyzing proteins from 10,000 primary CD4 T, CD8 T, NK, and B cells. The immune cell spectral assay library generated included 10,544 protein groups and 127,106 peptides. The proteomic profiles of 10,000 primary human immune cells obtained from 15 healthy volunteers analyzed using DIA revealed the highest heterogeneity of B cells among other immune cell types and the similarity between CD4 T and CD8 T cells. All data and spectral library are deposited in ProteomeXchange (PXD047742).

Rheumatoid arthritis (RA), a prevalent systemic autoimmune disease, affects 0.5–1% of the global population and is characterized by persistent joint inflammation and potential bone damage. Despite the utilization of Disease-Modifying Antirheumatic Drugs (DMARDs) and TNF inhibitors (TNFi) to manage RA, approximately one-third of patients do not response to these treatments, underscoring the urgent need for more precise therapeutic approaches. This study presents a proteomics-based machine learning approach to identify serum biomarkers capable of predicting individual patient responses to TNFi therapy, specifically infliximab By analyzing serum samples from 71 responders and 122 non-responders using Data-Independent Acquisition mass spectrometry (DIA-MS) for comprehensive proteomic profiling, we identified a panel of 10 multi-biomarkers, SAA2, MBL2, CLU, F5, FCGBP, IGFBP3, FGA, PROS1 and PCOLCE. These biomarkers are closely linked to key biological processes in RA, such as inflammation (SAA2, MBL2), immune modulation (CLU, FCGBP), coagulation (F5, PROS1, FGA), and tissue remodeling (PCOLCE). A logistic regression model utilizing these biomarkers achieved an accuracy of 82%, with a sensitivity of 0.74 and a specificity of 0.87. These biomarkers correlating with RA disease activity and patient response to infliximab, have the potential to enable a predictive model for personalized treatment. The advancement suggests a future shift towards a more predictive, personalized approach in RA management, potentially Improving by reducing the reliance on the current trial-and-error method in therapy selection.

Dysregulation of O-GlcNAcylation has emerged as a potential biomarker for several diseases, particularly cancer. The role of OGT (O-GlcNAc transferase) in maintaining O-GlcNAc homeostasis has been extensively studied; nevertheless, the regulation of OGA (O-GlcNAcase) in cancer remains elusive. Here, we demonstrated that the multifunctional protein RBM14 is a regulator of cellular O-GlcNAcylation. By investigating the correlation between elevated O-GlcNAcylation and increased RBM14 expression in lung cancer cells, we discovered that RBM14 promotes ubiquitin-dependent proteasomal degradation of OGA, ultimately mediating cellular O-GlcNAcylation levels. In addition, RBM14 itself is O-GlcNAcylated at serine 521, regulating its interaction with the E3 ligase TRIM33, consequently affecting OGA protein stability. Moreover, we demonstrated that mutation of serine 521 to alanine abrogated the oncogenic properties of RBM14. Collectively, our findings reveal a previously unknown mechanism for the regulation of OGA and suggest a potential therapeutic target for the treatment of cancers with dysregulated O-GlcNAcylation.

Irregularities in insulin signaling have significantly increased the risk of various cancers, yet the precise underlying mechanisms remain unclear. Within our study, we observed that inhibiting neddylation enhances cancer cell migration across different cancer types by activating both insulin receptor substrates 1 and 2 (IRS1 and IRS2), along with the PI3K/AKT signaling pathway. Notably, in the context of high-grade serous carcinoma (HGSC) patients, whether they had type 2 diabetes mellitus or not, IRS1 and IRS2 displayed a parallel relationship with each other while exhibiting an inverse relationship with NEDD8. We also identified C-CBL as an E3 ligase responsible for neddylating IRS1 and IRS2, with clinical evidence further confirming a reciprocal relationship between C-CBL and pAKT, thereby reinforcing the tumor suppressive role of C-CBL. Altogether, these findings suggest that neddylation genuinely participates in IRS1 and IRS2-dependent insulin signaling, effectively suppressing cancer cell migration. Thus, caution is advised when considering neddylation inhibitors as a treatment option for cancer patients, particularly those presenting with insulin signaling dysregulations linked to conditions like obesity-related type 2 diabetes or hyperinsulinemia.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammation in the synovial lining of the joints. Key inflammatory cytokines such as interleukin-6 (IL-6), TNF-α, and others play a critical role in the activation of local synovial leukocytes and the induction of chronic inflammation. Tocilizumab (TCZ), a humanized anti-IL-6 receptor monoclonal antibody, has demonstrated significant clinical efficacy in treating RA patients. However, similar to other inflammatory cytokine blockers, such as TNF-alpha inhibitors, Interleukin-1 inhibitors, or CD20 inhibitors, some patients do not respond to treatment. To address this challenge, our study employed a high-precision proteomics approach to identify protein biomarkers capable of predicting clinical responses to Tocilizumab in RA patients. Through the use of data-independent acquisition (DIA) mass spectrometry, we analyzed serum samples from both TCZ responders and non-responders to discover potential biomarker candidates. These candidates were subsequently validated using individual serum samples from two independent cohorts: a training set (N = 70) and a test set (N = 18), allowing for the development of a robust multi-biomarker panel. The constructed multi-biomarker panel demonstrated an average discriminative power of 86 % between response and non-response groups, with a high area under the curve (AUC) value of 0.84. Additionally, the panel exhibited 100 % sensitivity and 60 % specificity. Collectively, our multi-biomarker panel holds promise as a diagnostic tool to predict non-responders to TCZ treatment in RA patients.

As the number of prohibited drugs has been progressively increasing and analytical methods for detecting such substances are renewed continuously for doping control, the need for more sensitive and accurate doping analysis has increased. To address the urgent need for high throughput and accurate analysis, liquid chromatography with tandem mass spectrometry is actively utilized in case of most of the newly designated prohibited substances. However, because all mass spectrometer vendors provide data processing software that is incapable of handling other instrumental data, it is difficult to cover all doping analysis procedures, from method development to result reporting, on one platform. Skyline is an open-source and vendor-neutral software program invented for the method development and data processing of targeted proteomics. Recently, the utilization of Skyline has been expanding for the quantitative analysis of small molecules and lipids. Herein, we demonstrated Skyline as a simple platform for unifying overall doping control, including the optimization of analytical methods, monitoring of data quality, discovery of suspected doping samples, and validation of analytical methods for detecting newly prohibited substances. For method optimization, we selected the optimal collision energies for 339 prohibited substances. Notably, 195 substances exhibited a signal intensity increase of >110% compared with the signal intensity of the original collision energy. All data related to method validation and quantitative analysis were efficiently visualized, extracted, or calculated using Skyline. Moreover, a comparison of the time consumed and the number of suspicious samples screened in the initial test procedure highlighted the advantages of using Skyline over the commercially available software TraceFinder in doping control.