Improved Peptide Identification for Proteomic Analysis Based on Comprehensive Characterization of Electron Transfer Dissociation Spectra

Abstract

In recent years, electron transfer dissociation (ETD) has enjoyed widespread applications from sequencing of peptides with or without post-translational modifications to top-down analysis of intact proteins. However, peptide identification rates from ETD spectra compare poorly with those from collision induced dissociation (CID) spectra, especially for doubly charged precursors. This is in part due to an insufficient understanding of the characteristics of ETD and consequently a failure of database search engines to make use of the rich information contained in the ETD spectra. In this study, we statistically characterized ETD fragmentation patterns from a collection of 461 440 spectra and subsequently implemented our findings into pFind, a database search engine developed earlier for CID data. From ETD spectra of doubly charged precursors, pFind 2.1 identified 63-122% more unique peptides than Mascot 2.2 under the same 1% false discovery rate. For higher charged peptides as well as phosphopeptides, pFind 2.1 also consistently obtained more identifications. Of the features built into pFind 2.1, the following two greatly enhanced its performance: (1) refined automatic detection and removal of high-intensity peaks belonging to the precursor, charge-reduced precursor, or related neutral loss species, whose presence often set spectral matching askew; (2) a thorough consideration of hydrogen-rearranged fragment ions such as z + H and c -H for peptide precursors of different charge states. Our study has revealed that different charge states of precursors result in different hydrogen rearrangement patterns. For a fragment ion, its propensity of gaining or losing a hydrogen depends on (1) the ion type (c or z) and (2) the size of the fragment relative to the precursor, and both dependencies are affected by (3) the charge state of the precursor. In addition, we discovered ETD characteristics that are unique for certain types of amino acids (AAs), such as a prominent neutral loss of SCH 2 CONH 2 (90.0014 Da) from z ions with a carbamidomethylated cysteine at the N-terminus and a neutral loss of histidine side chain C 4 N 2 H 5 (81.0453 Da) from precursor ions containing histidine. The comprehensive list of ETD characteristics summarized in this paper should be valuable for automated database search, de novo peptide sequencing, and manual spectral validation.

Dissociation Channel Dependence on Peptide Size Observed in Electron Capture Dissociation of Tryptic Peptides

“…In this sense, the strong dependence of ion fragment types on peptide size should also be introduced in such models. Furthermore, the methodology presented in this work will now be applied to other fragmentation spectra, and most relevantly to ETD fragmentation: comparison of the ECD and ETD fragmentation behavior both for specific peptides and for entire fragmentation datasets [49] could lead to further knowledge on the similarities and differences between these two kin activation methods.…”

Human METTL20 Methylates Lysine Residues Adjacent to the Recognition Loop of the Electron Transfer Flavoprotein in Mitochondria

“…The fragmentation spectra of modified peptides were interpreted manually. ETD fragment ions were assigned as c and z ions (including related z +H ions) (42). The relative abundance of methylation of ETFβ was determined by analysis of an AspN-generated peptide (EPRYATLPNIMKAKKKKI) that contains both methylated lysine residues.…”

Activated Ion Electron Transfer Dissociation for Improved Fragmentation of Intact Proteins

“…31,32 Postreaction activation of ETnoD products with gentle collisions (termed ETcaD) produces more extensive and intense fragment ions from N−Cα backbone cleavages for peptide cations, but many fragments generated have skewed isotope distributions due to hydrogen abstractions. 29,33,34 Ideally then, activation would be applied concurrently with the ETD reaction; collisional activation of the precursor ions, however, requires resonant excitation during ETD, increasing the velocity of the ions and inhibiting ion−ion reactions. 35−37 Instead, we have introduced a viable alternative, IR photon (10.6 μm) irradiation concurrent to the ETD reaction, termed activated ion ETD (AI-ETD), and have shown the benefits it affords peptide fragmentation for shotgun analyses.…”