Internal Fragments Generated by Electron Ionization Dissociation Enhances Protein Top-down Mass Spectrometry

Abstract

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation (ECD) and electron transfer dissociation (ETD) are widely used for these types of analysis, however these fragmentation methods can be inefficient due to the low energy electrons fragmenting the protein without the dissociation products; that is no detection of fragments formed. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (> 20 eV) has been shown to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that do not contain the C-terminus or N-terminus of the protein. Conventionally, internal fragments have been disregarded as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ~20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ~50% to ~99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. By including internal fragments in the data analysis, previously unassigned peaks can be readily and accurately assigned to enhance the efficiencies of top-down protein sequencing experiments.

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

“…Additionally, the performance of traditional collision-based methods is frequently lackluster for the sequence characterization of intact proteins. Other ion activation methods such as electron-based dissociation 3,4,15 and ultraviolet photodissociation (UVPD) 5,14,16−19 have been shown to achieve more efficient fragmentation, resulting in higher sequence coverages, but the large number of still unassigned peaks remains an issue. All of these ion activation methods generate internal fragment ions, products that do not contain either the N-or C-termini of the protein.…”

“…20 Recently, curation of internal fragments has been given more attention and has been shown to improve sequence coverage of proteins when properly assigned. 15,20,26−28 These initial studies focused on the production of internal fragment ions by CID, 20,26,27 electron ionization dissociation (EID), 15,26,27 and 213 nm UVPD. 25,26 The latter method, UVPD, might be expected to be especially prone to extensive generation of internal fragment ions because it is a high-energy activation method that produces a variety of fragment ion types (a/x, b/ y, and c/z and variations thereof).…”

Improving the Center Section Sequence Coverage of Large Proteins Using Stepped-Fragment Ion Protection Ultraviolet Photodissociation

“…One particularly daunting challenge is generating and interpreting MS/MS spectra of very large proteins, a problem that requires sophisticated data processing and informatics algorithms and requires ion activation methods that are complementary to or more effective than conventional collisional-based methods. Other ion activation techniques such as electron capture dissociation (ECD), 3 electron transfer dissociation (ETD), 4 electron ionization dissociation (EID), 5 activated ion electron transfer dissociation (AI-ETD), 6 and ultraviolet photodissociation (UVPD) 7−11 have been shown to increase the number of diagnostic fragment ion identifications. However, as proteins increase in size, identifying fragment ions becomes even more difficult as a result of increased spectral congestion 12 and decreased fragment ion signal-to-noise, 4 two factors that have slowed the widespread adoption of top-down methods.…”

“…Recently software has been developed allowing the identification of internal fragment ions. 5,40,41 Notably, inclusion of an internal fragment has been shown to increase and expand sequence coverage in the center section of proteins for a variety of dissociation methods. 5,40−42 However, including internal fragments in searches may result in a greater chance of false positive identifications, and in some applications it is advantageous to minimize secondary dissociation to minimize the production of internal ions and reduce spectral congestion.…”