Improving the Sensitivity and Selectivity of Laser Desorption Ionization Using N-Heterocyclic Carbene Mass Ionization Tags

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.

Grant Number: 5R21GM155773-02
NIH Institute/Center: NIH
Principal Investigator: Jon Camden