Next-generation approaches for comprehensive mapping of post-translational modification landscapes

SUMMARY The precise detection of post-translational modifications (PTMs) is essential for understanding the complex regulatory networks that dictate protein function, cellular signaling, and disease progression. While advances in mass spectrometry, proteomic technologies, and bioinformatics have greatly expanded the repertoire of known PTMs, a comprehensive and systematic elucidation of their biological roles remains challenging. A major bottleneck lies in the lack of highly specific tools for enriching the modified proteome, limiting our capacity to fully explore and characterize these dynamic modifications. Our overarching goal is to overcome these barriers by developing next-generation detection strategies that surpass the limitations of current affinity reagents, such as antibodies, which often lack the specificity to discriminate among closely related PTMs, the sensitivity to detect low-abundance modifications, or the ability to analyze and modulate PTM activity in living cells. To tackle these issues, our laboratory combines high- throughput experimental platforms with computational design approaches to obtain novel reagents capable of resolving subtle differences among modification states and tracking PTMs in real time. We will pursue two primary lines of investigation: first, we will engineer families of highly specific PTM- binding domains (reader domains) for PTMs recognition and detection. This approach already enabled us to enhance their affinity while preserving their inherent specificity, enabling the generation of robust probes for unparalleled profiling of tyrosine-phosphorylated proteomes and genome-wide chromatin methylation. We will expand this strategy to additional reader domains recognizing different PTMs, thereby broadening the range of PTMs that can be analyzed or allowing the same PTM to be interrogated across different cellular substrates and compartments. Second, we will advance functional understanding of PTMs in biological and disease contexts, by integrating engineered reader domains into proteomic and bioinformatic workflows to enable the simultaneous detection of multiple PTMs in complex samples with minimal background noise. Panels of high-affinity engineered reader domains capable of recognizing diverse PTMs will be fused to covalent protein–peptide pairs we have developed, creating a one-pot affinity matrix for massively parallel PTM detection. This strategy will allow us to analyze PTM crosstalk and its regulatory roles in cellular signaling with unprecedented efficiency and scalability. Additionally, by coupling engineered domains with unique DNA barcodes, we will develop a novel method, PTM-grab&seq, to simultaneously quantify hundreds of PTMs through next-generation sequencing of the associated oligonucleotides. These strategies will provide transformative tools for creating comprehensive PTM landscape maps and reveal how modifications interact and fluctuate in response to environmental or pathological changes, offering valuable insights into cellular regulation and disease mechanisms. Project Number: 1R35GM162512-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Gianluca Veggiani | Institution: LOUISIANA STATE UNIV A&M COL BATON ROUGE, BATON ROUGE, LA | Award Amount: $375,000 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MCST-G (56)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11272179