Successfully Identifying Proteins and Their Modifications Using Electron Transfer Dissociation Linear Ion Trap Mass Spectrometry

March 1, 2009

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The authors discuss the use of electron transfer dissociation as a reliable tool in proteomic research, especially in conjunction with a linear ion trap, for sequence analysis of post-translationally modified and highly basic peptides.

Electron transfer dissociation (ETD) with linear ion trap mass spectrometry is a tool with significant advantages for sequence analysis of post-translationally modified and highly basic peptides. Traditionally, collision-activated dissociation (CAD) has been used to identify proteins and to attempt to identify and locate the site of their modifications; however, this technique has specific limitations, which will be discussed in this article. The utility of ETD as a reliable tool in proteomic research will be illustrated, especially in conjunction with a linear ion trap, by readily identifying peptides resistant to analysis by CAD. Although it is a relatively new peptide/protein fragmentation method, ETD holds great promise to advance the field of protein identification via mass spectrometry.

Post-translational modification (PTM) is the chemical modification of a protein after its translation and is one of the later steps in protein biosynthesis. The analysis of proteins and their PTMs is particularly important for the study of many diseases, including cancer, diabetes, cardiovascular disease, and neurodegenerative disorders such as Alzheimer's disease. This is because during or after protein synthesis, a variety of covalent protein modifications can occur. These modifications are needed for normal cellular function. However, alterations in the regulation of these modifications can lead to diseases such as Alzheimer's disease, cancer, and erectile dysfunction. Protein modification can enhance or decrease protein activity, allow interaction with other proteins, and localize protein to a specific place in the cell.

PTMs such as phosphorylation, acetylation, and methylation are used as chemical switches to activate or inactivate the histone regulation of gene transcription, DNA replication, and DNA damage repair. Histones are the chief protein components of chromatin. They act as spools around which DNA winds, and they play a role in gene regulation. Identifying such PTMs is essential because it leads to insights into the function and role of the protein in the biological system.

Identifying Proteins with Collisionally Activated Dissociation

Mass spectrometry has played an integral role in the identification of proteins and their PTMs. Collisionally activated dissociation (CAD) is a common technique used to analyze proteins by mass spectrometry. An enzyme (typically trypsin) is used to digest the proteins into smaller peptides, which are then separated by reversed phase chromatography. The peptides are then introduced into a mass spectrometer by electrospray ionization (ESI), and sequence information is obtained by tandem mass spectrometry (MS-MS). Peptides ionize via ESI to form peptide cations with a number of charge states for which the lower charge states are optimal for CAD analysis. Low-energy CAD tandem MS has been the most common method used to dissociate peptide ions for subsequent sequence analysis.

Figure 1

Analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with CAD because the modification is usually labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence and phosphorylation site information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD MS.

Depending on the protein sequence, sometimes trypsin can generate peptides that are too small or too large for efficient analysis. In those situations, the lack of a sufficiently diverse set of fragment ions does not allow confident sequence analysis. Therefore, CAD is most effective for shorter, low-charged peptides. This widely used approach limits investigators to the analysis of a subset of all available peptides. This can also prevent the detection of multiple PTMs and the understanding of their biological function.

An Advanced Fragmentation Technique: Electron Transfer Dissociation

Electron transfer dissociation (ETD) is a new method of fragmenting peptides that uses ion–ion gas-phase chemistry. ETD fragments peptides using chemical energy by transferring an electron from a radical anion to a protonated peptide. This induces fragmentation of the peptide backbone. ETD produces peptide backbone sequence and peptide side-chain information that is often complementary to CAD.

Figure 2

Compared with the conventional CAD technique, ETD offers a more robust method of characterizing PTMs and interrogating large peptides or even whole proteins. ETD produces product ions from peptides containing common PTMs, peptides with multiple basic residues, and intact proteins amenable to peptide interpretation. ETD can also readily fragment peptides containing disulfide bonds. ETD fragmentation produces many more product ions amenable to peptide interpretation than CAD fragmentation methodology.

ETD is a variation of a similar fragmentation technique developed for Fourier transform–ion cyclotron resonance (FT-ICR) instruments. The use of an electron-bearing transfer reagent instead of a free electron to affect peptide fragmentation enables the implementation of ETD in the widely used RF quadrupole ion traps. RF ion trap mass spectrometers are low-cost, low maintenance, and widely accessible compared to FT-ICR-MS systems.

ETD has been implemented in linear ion traps as well as their predecessor 3D ion traps. Although the implementation of ETD in 3D traps provides unique benefits over CAD alone, this combination does not provide all the technical capabilities required for proteomics analyses. With ETD on a nonlinear ion trap, it has not been possible to control the crucial fragmentation process well or to analyze larger peptides due to the inherently limited ion capacity of 3D traps. In response, researchers have made the ETD capability available on a linear ion trap (Thermo Scientific LTQ XL mass spectrometer).

Figure 3

ETD on a linear ion trap provides a powerful tool for the identification of proteins and their PTMs. The LTQ XL linear ion trap mass spectrometer delivers more structural information than any other ion trap, and the ETD option provides sequence information not available from conventional methods. The differentiating feature of ETD on a linear ion trap compared with nonlinear ion traps is how the ion–ion reaction takes place. While the ETD functionality is completely automated and usually requires no further user intervention, the linear ion trap provides users with more complete control because the ions can be controlled independently via software, allowing the ion populations to be manipulated if necessary. A linear ion trap mass spectrometer has the ability to process a high number of samples and analyze both large and small molecules at low levels of concentration. With a nonlinear ion trap the process is much more complex and time-consuming.

Application Example

In a recent application (1), highly basic peptides and a number of biologically important PTMs were analyzed by CAD and ETD linear ion trap MS. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions, thus extensive peptide sequence information.

The sensitivity and robustness of ETD is essential to proteomic analysis. ETD offers a highly reliable solution that is extremely user friendly, requires little day-to-day maintenance, and delivers highly accurate data. It is also much easier to perform data analysis with ETD because complementary data processing software is available.


The application of ETD in proteomic research shows great promise for studying the mechanism of diseases such as cancer, for drug discovery studies, and for characterizing cellular function and signaling. ETD will expand current analysis to include more basic, nontryptic peptides and proteins, which will enable the identification of various PTMs within the context of each other as well as potentially identify new protein isoforms.

ETD coupled with linear ion trap MS is opening the door to the widespread implementation of ETD for various proteomic applications. ETD on linear ion traps continues to drive the field of proteomics and has proven to be an effective alternative to CAD and ETD on nonlinear traps for peptide sequence analysis. In the future, ETD on linear ion traps is predicted to become the fragmentation technique of choice.


(1) Leann M. Mikesh et al., Biochemica et Biophysica Acta (2006), doi:10.1016/j.bbapap.2006.10.003