Comparison of the Electron Capture Dissociation Fragmentation Behavior of Doubly and Triply Protonated Peptides from Trypsin, Glu-C, and Chymotrypsin Digestion Anastasia Kalli and Kristina Håkansson* Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055 Received January 17, 2008
In bottom-up proteomics, proteolytically derived peptides from proteins of interest are analyzed to provide sequence information for protein identification and characterization. Electron capture dissociation (ECD), which provides more random cleavages compared to “slow heating” techniques such as collisional activation, can result in greater sequence coverage for peptides and proteins. Most bottomup proteomics approaches rely on tryptic doubly protonated peptides for generating sequence information. However, the effectiveness, in terms of peptide sequence coverage, of tryptic doubly protonated peptides in ECD remains to be characterized. Herein, we examine the ECD fragmentation behavior of 64 doubly- and 64 triply protonated peptides (i.e., a total of 128 peptide ions) from trypsin, Glu-C, and chymotrypsin digestion in a Fourier transform ion cyclotron resonance mass spectrometer. Our findings indicate that when triply protonated peptides are fragmented in ECD, independent of which proteolytic enzyme was used for protein digestion, more c- and z-type product ions are observed, and the number of complementary fragment pairs increases dramatically (44%). In addition, triply protonated peptides provide an increase (26%) in peptide sequence coverage. ECD of tryptic peptides, in both charge states, resulted in higher sequence coverage compared to chymotryptic and Glu-C digest peptides. The peptide sequence coverage we obtained in ECD of tryptic doubly protonated peptides (64%) is very similar to that reported for electron transfer dissociation of the same peptide type (63%). Keywords: FTICR • Fourier transform ion cyclotron resonance • MS • mass spectrometry • CAD • collision activated dissociation • ECD • electron capture dissocation • ETD • electron transfer dissociation MS/MS • tandem mass spectrometry
Introduction Mass spectrometry has become the method of choice for protein identification, characterization and quantification due to its high accuracy and sensitivity that allow analysis of lowabundance proteins and also its ability to analyze complex protein samples from cells, tissues and organisms.1–4 Bottomup5–10 and top-down11–13 approaches are both used in mass spectrometric protein analysis, with the former being the most widely applied. In the bottom-up approach, proteins are digested with a sequence-specific protease and the derived proteolytic peptides are analyzed by tandem mass spectrometry to provide sequence information for protein identification. In tandem mass spectrometry, individual peptides are first selected and then fragmented by, for example, collisions with an inert gas. Preferably, a single fragmentation technique should result in the formation of a sufficient number of product ions to unambiguously identify a peptide and, subsequently, the corresponding protein in a database search, either based on MS/MS data from a single peptide or from multiple peptides from the same protein. However, in cases where genome data * To whom correspondence should be addressed. E-mail,
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2834 Journal of Proteome Research 2008, 7, 2834–2844 Published on Web 06/13/2008
are not available to generate a protein database, or when peptides are post-translationally modified, de novo sequencing may be required. De novo sequencing by mass spectrometry remains a challenge because it requires cleavage between each pair of amino acid residues and detection of all generated products. Collision activated dissociation (CAD),14,15 which induces cleavage of peptide backbone amide bonds to generate Nterminal b-type ions and C-terminal y-type ions, is by far the most widely used technique for obtaining peptide sequence information by MS/MS. However, in some cases, CAD results only in uninformative neutral losses or selective cleavage at specific residues, thereby preventing formation of a sufficient number of product ions for peptide identification.16,17 Electron capture dissociation (ECD)18,19 and electron transfer dissociation (ETD),20 which result in extensive cleavage of peptide backbone N-CR bonds to form N-terminal c-type and Cterminal z-type product ions, are complementary to CAD and constitute promising methods for de novo sequencing of peptides and proteins. In ECD, precursor ions are irradiated with low energy (
