Internal Fragments Generated from Different Top-Down Mass Spectrometry Fragmentation Methods Extend Protein Sequence Coverage

Abstract

Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N-or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photonbased fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome c, myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15−20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.

Influence of Primary Structure on Fragmentation of Native-Like Proteins by Ultraviolet Photodissociation

“…FDR for each mass-shifted data set of each of the three proteins is shown in Figure S1. Although internal fragments are expected and present in UVPD mass spectra, , only terminal fragments were considered in the present study owing to challenges with confidently assigning internal fragment ions that require high mass accuracy and special considerations to resolve assignment ambiguity. − …”

“…Alternatively, backbone cleavages in the midsection of the protein may occur with high efficiency, but the resulting fragment ions decompose into smaller ions or internal ions or may produce ions that fall in highly congested regions of the mass spectra, rendering them ineffectively deconvoluted and unassignable. These latter explanations have generated considerable speculation − , and motivated recent efforts to resolve them by the development of new search algorithms ,, and experimental strategies such as proton transfer reactions , and fragment ion protection. , …”

“…FDR for each massshifted data set of each of the three proteins is shown in Figure S1. Although internal fragments are expected and present in UVPD mass spectra, 67,68 only terminal fragments were considered in the present study owing to challenges with confidently assigning internal fragment ions that require high mass accuracy and special considerations to resolve assignment ambiguity. 65−70 Although a and a+1 were both the most frequent and the most abundant sequence fragments, each of the other fragment types was identified at generally comparable frequencies and abundances.…”

Internal Fragment Ions Disambiguate and Increase Identifications in Top-Down Proteomics

“…Previous work suggests that a large fraction of these unidentified fragment ions can be attributed to internal fragments. − Unlike canonical terminal fragments, which contain either the original intact proteoform N- or C-terminus, internal fragments originate from proteoform molecules that have been fragmented multiple times and no longer possess either of the original termini. Several top-down analyses of individual proteins have been published and demonstrate the utility of internal fragments for understanding proteoform fragmentation spectra and improving sequence coverage. − Despite the demonstrated utility of internal fragments, − they are not typically used in top-down analyses.…”

“…Previous work suggests that a large fraction of these unidentified fragment ions can be attributed to internal fragments. − Unlike canonical terminal fragments, which contain either the original intact proteoform N- or C-terminus, internal fragments originate from proteoform molecules that have been fragmented multiple times and no longer possess either of the original termini. Several top-down analyses of individual proteins have been published and demonstrate the utility of internal fragments for understanding proteoform fragmentation spectra and improving sequence coverage. − Despite the demonstrated utility of internal fragments, − they are not typically used in top-down analyses. The addition of internal fragment ions to the search space can cause a computational explosion in theoretical fragment ions and negatively impact the false discovery rate (FDR) of the experiment. − Although sequence coverage for identified proteoforms improves with the addition of internal fragments, the number of confident proteoform identifications decreases .…”