PROJECT SUMMARY Even two decades after the complete sequencing of the human genome, a significant subpopulation (~10%) of the encoded proteins remains unknown. The questions surrounding this “dark” proteome are further compounded because there is not a one-to-one correspondence between genes and proteins and the function of expressed proteins can be changed by hundreds of different types of post-translational modifications. The resulting protein variants, or proteoforms, could be in the millions and may appear at concentrations well below the detection limit of current methods. Mass spectrometry (MS) is one of most powerful tools in the modern proteomics toolbox; however, MS only measures things that can be ionized and promoted to the gas phase, highlighting the importance of the ionization process. Today, measurement of high-abundance proteins is a trivial task for modern mass spectrometers, but closer examination reveals that low-abundance proteoforms comprising the “dark” proteome remain elusive. Therefore, new measurement capabilities are urgently needed that can increase the sensitivity and selectivity of the ionization process. While improvements in MS instrumentation are expected to continue, a parallel approach relying on chemical methods to increase sensitivity would provide a synergistic route to detect low abundance proteoforms. Here, we outline a high-risk but high- reward strategy for improving the ionization efficiency of these low abundance peptides and post-translationally modified peptides. Specifically, proof-of-concept experiments will demonstrate that N-heterocyclic carbene (NHC) decorated gold nanoparticles (AuNPs) are an excellent platform for the selective capture and fragment- free ionization of peptides, delivering at least two orders of magnitude improvement over state-of-the-art methods. Additionally, we will demonstrate that these NHC-AuNPs can improve the detection of post- translationally modified proteins and are compatible with pre-existing proteomics workflows employing bioorthogonal click chemistries. Lastly, while the proof-of-concept studies proposed here target bottom-up MS proteomics applications, the NHC mass tag platform is quite general and would have broader implications for MS applications ranging from tissue and single-cell imaging to disease biomarker identification and detection.

PROJECT NARRATIVE The identification and quantification of low-abundance and post-translationally modified proteins is a formidable and unmet challenge with tremendous potential to impact human health. The proposed research will develop a new chemical platform employing N-heterocyclic carbenes and gold nanoparticles to improve the ionization efficiency of peptides, which is critical for elucidating protein structure. This increased sensitivity has the potential to reveal new disease biomarkers and provide a protein-level view of cellular functions.