Investigating the Efficiency of Ultraviolet Photodissociation in Peptides Modified with <i>N</i> -Terminal UV-Absorbing Chromophores

Ultraviolet photodissociation (UVPD) has emerged as a powerful alternative to conventional collision-induced dissociation (CID) for peptide and protein sequencing in mass spectrometry-based proteomics. However, the UVPD efficiency depends on the presence of UV-absorbing chromophores within analytes. Here, we systematically investigate how <i>N</i>-terminal modification of peptides with aromatic chromophores influences the 193 nm UVPD efficiency and fragmentation behavior. Using NHS ester chemistry, human cell lysate digests were derivatized with a range of UV-absorbing aromatic labels and analyzed on an Omnitrap-Orbitrap platform equipped with a 193 nm ArF excimer laser. Compared with unmodified controls, derivatized peptides displayed a modestly increased fragmentation efficiency and a greater diversity of sequence-informative fragments, particularly <i>b</i>-type ions. Peptides lacking aromatic amino acids benefited most from N-terminal aromatic labeling, exhibiting enhanced fragmentation and sequence coverage. These findings demonstrate that incorporation of aromatic chromophores at the N-terminus can enhance UVPD performance and improve spectral interpretability, thereby expanding the applicability of UVPD in bottom-up proteomics workflows.