Mechanistic Insights into Intramolecular Gas-Phase Crosslinking of Peptides and Carbene or Nitrile Imine Intermediates

Abstract

This dissertation presents a comprehensive study on the use of photochemical crosslinking, advanced tandem mass spectrometry, and computational simulations to investigate noncovalent interactions and crosslink- ing behaviors in gas-phase peptide ions. The work combines experimental and theoretical approaches to analyze how different crosslinking chemistries, protonation sites, amino acid compositions, and scaffold stereochemistries affect peptide structure and reactivity, providing a new framework for understanding the fundamental interactions of intramolecular peptide sequences with various phototags in gas-phase environments, advancing the application of mass spectrometry as a powerful tool for biomolecular structural analysis.Chapter 1 introduces the principles of mass spectrometry, including ionization, mass analysis, and detection methods, enabling the characterization of biomolecules with minimal sample preparation. This chapter also discusses photochemical crosslinking as a method for probing noncovalent interactions, focusing on the role of reactive intermediates such as nitrenes, carbenes, and nitrile imines, providing insights into three-dimensional structures and molecular interactions, particularly for complex biomolecular assemblies. In Chapter 2, the efficacy of carbene crosslinkers activated by diazirine under 355 nm is examined using peptide scaffolds (s-LAAG, s-ALAG, and s-AALG). Crosslinking yields were consistent across these scaffolds, demonstrating a robust crosslinking mechanism that is independent of the sequence order of alanine and leucine residues. Hydrogen-deuterium exchange, carboxyl C-terminus blocking, and analysis of CID-MSn spectra of reference synthetic products revealed that a significant fraction of crosslinks involved the Gly amide and carboxyl groups. Contact analysis of long Born-Oppenheimer molecular dynamics (BOMD) trajectories was used to count close contacts between the incipient carbene and peptide atoms, and further provided insights into the thermal behavior of peptide ions, validating the s-AALG scaffold as a robust model for carbene crosslinking studies in hydrophobic environments. Chapter 3 extends the analysis to peptide sequences containing basic residues: proline and histidine, within s-AAPG and s-AAHG scaffolds. Crosslinking yields were significantly reduced in these peptides, attributed to the unique protonation and conformational characteristics of the basic residues. Detailed BOMD simulations revealed that proline forms a stable hydrogen-bond network and affects crosslinking patterns, while histidine’s protonation at the imidazole group introduces structural constraints. These findings under- score the role of amino acid basicity and protonation in governing crosslinking efficiency and specificity. In Chapter 4, nitrile imine intermediates generated by photodissociation of tetrazole-tagged peptide conjugates on cyclohexane scaffolds with distinct stereochemistry (cis-1,2- and trans-1,4-cyclohexane) is introduced. Despite expected steric hindrance for the trans-1,4 configuration, crosslinking occurs in both scaffold types, challenging conventional stereochemical assumptions. High-resolution cyclic ion mobility mass spectrometry, BOMD, and density functional theory (DFT) calculations allow us to match theoretical and experimental collision cross sections, providing insights into stereochemical effects on crosslinking yields and attachment sites. These findings reveal that the flexibility of the cis scaffold facilitates interactions, while the trans scaffold imposes conformational constraints that affect crosslinking efficiency. Chapter 5 focuses on the reactivity of peptides with C-terminal lysine or arginine residues when crosslinked with nitrile imine intermediates. Experiments showed that lysine primarily crosslinks through the carboxyl group, while arginine’s guanidine group exhibits unique reactivity, likely due to proton transfer steps that precede C–N bond formation. Computational Gibbs energy calculations reveal this reaction as an endothermic proton transfer followed by an exothermic bond formation step, highlighting a novel mechanism in gas-phase peptide ion chemistry that has not been observed in condensed phases. Ion mobility data allowed for accurate comparisons of experimental and theoretical collision cross sections, offering a deeper understanding of ion structures and reactivity.