High Throughput Peptide Interaction Discovery, Epitope Mapping and “Split” proximity labelling
Biological and Biomimetic Chemistry
Organic Molecular Chemistry - Synthesis and Characterisation
Pharmacology
Final Report Abstract
In human cells, nearly a fourth of actively translated peptides comes from previously unannotated long noncoding RNA, with no known functional roles. Additionally, the role of short peptides, generated through maturation of proteins translated from canonical genes, has remained largely elusive. This substantially untamed microcosm offers untapped potential for devising novel pharmacological targets and understanding the underlying biology. We aimed at investigating these uncharted interactions using proximity labeling photocatalytic technology, μMap, towards high-throughput discovery of protein interactor and localization of reported, yet cryptic, 20-100 amino acid peptides. We initially sought to leverage affinity-based isolation of labeled peptides with reversible desthiobiotin (DTB)/streptavidin binding, but an initial assessment of this approach revealed unsurmountable challenges: (1) endogenous biotin background, (2) required large material inputs, (3) week-long sample processing, (4) cost of streptavidin-based sorbent materials. We surmised that, compared to liquid phase affinity-based peptide enrichment prior to MS analysis, a gas phase enrichment approach on ionized peptides could (1) minimize sample processing, (2) eliminate dilute analyte solutions/sample losses, (3) be integrated existing mass spectrometer technologies for simultaneous enrichment and analysis. With these criticalities in mind, we created a covalent modification capable of significantly changing the ion mobility of tryptic peptides in bottom-up proteomics, with minimal impact on its mass, to enable enrichment in the gas phase (timShift). We extensively optimized the molecular scaffold to yield a dicationic, aerodynamic, cysteine-reactive reagent increases the ion mobility of labeled peptides, physically separating them from unmodified peptides with similar m/z and enabling their targeted sequencing and quantification in whole proteomes. As proof-of-concept, we profiled >8,200 reactive cysteine sites, demonstrated a 20-fold increase in sensitivity vs. desthiobiotin/streptavidin enrichment, performed activity-based protein profiling of covalent fragments and selective electrophiles (e.g., sulfopin, a selective PIN1 covalent inhibitor) in a 96-well plate format, and successfully profiled cysteine reactivity with as few as 20,000 cells/sample. This novel technology proved to be a promising drop-in replacement for DTB/streptavidin affinity enrichment, and the Geri lab is currently applying this workflow towards short peptide interaction discovery using μMap, as well as other numerous collaboration projects at Weill Cornell Medicine.
