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.
Ultraviolet photodissociation (UVPD) mass spectrometry has gained attention in recent years for its ability to provide high sequence coverage of intact proteins. However, secondary dissociation of fragment ions, in which fragment ions subjected to multiple laser pulses decompose into small products, is a common phenomenon during UVPD that contributes to limited coverage in the midsection of protein sequences. To counter secondary dissociation, a method involving the application of notched waveforms to modulate the trajectories of fragment ions away from the laser beam, termed fragment ion protection (FIP), was previously developed to reduce the probability of secondary dissociation. This, in turn, increased the number of identified large fragment ions. In the present study, FIP was applied to UVPD of large proteins ranging in size from 29 to 55 kDa, enhancing the abundances of large fragment ions. A stepped-FIP strategy was implemented in which UVPD mass spectra were collected using multiple different amplitudes of the FIP waveforms and then the results from the mass spectra were combined. By using stepped-FIP, the number of fragment ions in the midsections of the sequences increased for all proteins. For example, whereas no fragment ions were identified in the middle section of the sequence for glutamate dehydrogenase (55 kDa, 55+ charge state), 10 sequence ions were identified by using UVPD-FIP.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.