Impact of Internal Fragments on Top-Down Analysis of Intact Proteins by 193 nm UVPD.

193 nm ultraviolet photodissociation (UVPD) allows high sequence coverage to be obtained for intact proteins using terminal fragments alone. However, internal fragments, those that contain neither N- nor C- terminus, are typically ignored, neglecting their potential to bolster characterization of intact proteins. Here, we explore internal fragments generated by 193 nm UVPD for proteins ranging in size from 17-47 kDa and using the ClipsMS algorithm to facilitate searches for internal fragments. Internal fragments were only retained if identified in multiple replicates in order to reduce spurious assignments and to explore the reproducibility of internal fragments generated by UVPD. Inclusion of internal fragment improved sequence coverage by an average of 18% and 32% for UVPD and HCD, respectively, across all proteins and charge states studied. However, only an average of 18% of UVPD internal fragments were identified in two out of three replicates relative to the average number identified across all replicates for all proteins studied. Conversely, for HCD, an average of 63% of internal fragments were retained across replicates. These trends reflect an increased risk of false-positive identifications and a need for caution when considering internal fragments for UVPD. Additionally, proton-transfer charge reduction (PTCR) reactions were performed following UVPD or HCD to assess the impact on internal fragment identifications, allowing up to 20% more fragment ions to be retained across multiple replicates. At this time, it is difficult to recommend the inclusion of the internal fragment when searching UVPD spectra without further work to develop strategies for reducing the possibilities of false-positive identifications. All mass spectra are available in the public repository jPOST with the accession number JPST001885.