Akira Tsuboyama

Electron-capture dissociation (ECD) has been shown to be a valuable tool for peptide analysis and protein identification, as well as for the characterization of protein post-translation modifications. ECD has several inherent advantages over traditional collision-induced dissociation (CID) that make it exceptionally useful for such studies. It tends to restrict cleavage to N-C(alpha) bonds along the peptide backbone, leaving post-translational modifications intact and available for mass spectral evaluation. Also, it performs especially well at fragmenting long peptides. However, traditionally there have been limitations to using ECD in protein and peptide analysis. Historically, it was restricted to expensive, complex, and resource-intensive Fourier-transform ion cyclotron resonance instruments. Additionally, the ECD reaction in these instruments occurred on a relatively long timescale, precluding its use in liquid chromatography–mass spectrometry experiments. Finally, some peptides simply were not amenable to ECD analysis for reasons not well understood. Here we describe the use of a novel ECD device, QuECD, that can be interfaced to a linear ion trap time-of-flight mass spectrometer, which addresses some of these weaknesses. The reaction time of this ECD technique is on par with traditional CID reactions and is compatible with the chromatographic timescale. In addition, we demonstrate "hot" ECD (HECD), a technique in which the energy of the electron beam used in the QuECD fragmentation reaction is raised to roughly an order of magnitude over traditional QuECD, promoting the fragmentation of otherwise difficult samples. We demonstrate both of these extensions to traditional ECD in an investigation of human transferrin.

Protein and peptide analysis via tandem mass spectrometry (MS-MS) has resulted in a wealth of information regarding protein identification, structure, and abundance levels over the past 10 years. Techniques such as neutral loss scanning and collision-induced dissociation (CID) have been especially helpful in facilitating the identification of a multitude of previously unknown sites of protein phosphorylation. However, many of the techniques used to obtain this information are labor intensive and work inconsistently. To address this problem, much effort has been put forth to find alternative methods of fragmenting peptides and proteins that are less difficult and applicable to a wide gamut of peptide classes. Examples of recently developed dissociation techniques include infrared multiphoton dissociation (IRMPD) and electron transfer dissociation (ETD). The implementation of these new techniques has widened the spectrum of peptides amenable to tandem mass spectral analysis. 

Electron-capture dissociation (ECD) is yet an additional peptide fragmentation technique that is gaining in popularity (1). ECD has a number of advantages over traditional fragmentation techniques such as CID. CID tends to cleave the bond between a posttranslation modification (PTM) and its partner peptide before it cleaves the peptide backbone bonds themselves. This premature loss of a PTM during the dissociation process precludes any ability to obtain structural information regarding that PTM. ECD, conversely, tends to leave the bond between a PTM and its partner peptide intact. Instead, it almost universally cleaves the N-Cα bonds of the peptide backbone (except for the N-terminal side of proline). This distinct dissociation pattern makes ECD an extremely useful technique for characterizing PTMs (2).  

In addition, ECD has proven to be effective at fragmenting very large peptides, and even small proteins. Indeed, ECD has been  applied successfully to ubiquitin, a 76-amino acid protein with a mass of 8564.47 Da. The experiment resulted in a sequence coverage of 85% at the amino acid level (65 of the 76 possible fragments were observed) (3).  

While ECD has been an effective method for peptide fragmentation for almost 10 years, historically it has been restricted to costly and complex Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometers, limiting its accessibility to a few specialized laboratories. In addition, the ECD reaction on these instruments occurs on a timescale that is too long to be coupled to a liquid chromatographic separation. Rather, samples must be introduced via direct infusion, prohibiting the study of complex mixtures.  

Recently, both of these issues have been resolved with the advent of QuECD, an ECD system that can be coupled to a time-of-flight mass analyzer (4). The QuECD cell comprises a radio frequency linear ion trap that is preceded by a stand-alone linear ion trap and succeeded by a time-of-flight mass analyzer (Figure 1). The initial ion trap accumulates ions of a specific mass and injects them into the QuECD cell, where the ECD reaction occurs. The ions are subsequently transported to the TOF analyzer for high resolution and mass accuracy analysis.  

 Figure 1: A schematic diagram of the QuECD platform. Specific ions are accumulated and trapped in the initial linear ion trap and directed to the RF-linear ion trap QuECD cell. The resulting fragments are then directed to the time-of-flight tube for high resolution and mass accuracy analysis. 

The QuECD system has two prominent advantages over traditional ECD. First, the QuECD reaction occurs on the chromatographic timescale, and is thus capable of analyzing complex mixtures via LC–ECD analysis. The second advantage of the QuECD system is rooted in the fact that the dissociation reaction is achieved by the action of an electron beam focused on the sample within the QuECD cell. Typically, the energy of this electron beam is <1 eV. However, not all peptides fragment efficiently at this energy level. With QuECD, the energy of the electron beam can be modulated to a level that more effectively fragments a particular peptide. "Hot" ECD (HECD) typically is performed at energy levels of 7–10 eV. Some peptides that are not amenable to fragmentation at lower energy levels exhibit very effective fragmentation under HECD conditions. 

In this work, we demonstrate examples of both of these extensions to conventional ECD in an analysis of human transferrin. First, we demonstrate QuECD's capability to perform an LC–ECD analysis on a Lys-C transferrin digest. Second, we show the effectiveness of HECD in getting optimal ECD fragmentation of specific transferrin glycopeptides. While these extensions to conventional ECD do not solve all peptide fragmentation problems, they add more tools to the biochemist's arsenal of techniques with which to characterize protein structure, function, and identity. 

Human transferrin (Sigma, St. Louis, Missouri) was resuspended in 8 M urea, 50 mM ammonium bicarbonate, pH 8.5, and subsequently incubated with 10 mM DTT for 60 min at 37 °C, then 20 mM iodoacetamide for 60 min at room temperature. The sample was diluted 10-fold in 50 mM ammonium bicarbonate, pH 8.5, Lys-C was added in a 1:100 Lys-C to protein ratio, and the solution was incubated overnight at 37 °C. 

Figure 2: Total ion chromatograms of the (a) LCâCID and (b) LCâECD analyses of a human transferrin Lys-C digest. 

The resulting peptides were separated on a 150 mm × 0.05 mm MonoCap C18 column (GL Sciences, Tokyo, Japan) using a 2–35% acetonitrile gradient over 120 min. Figure 2 shows total ion chromatograms for both a traditional LC–MS experiment (utilizing standard CID) and for an LC–ECD experiment. The similarity in the traces indicates that the fundamental change in the tandem MS mode does not alter the basic ability of the instrument to analyze peptides as they are eluted from the column. Of more interest is the similarity between the base peak intensities of the MS-MS events during the chromatographic run (Figure 3). This similarity attests to the ability of QuECD to work on the chromatographic timescale. In fact, the scan speed observed during ECD analysis is only marginally slower than that observed during traditional CID analysis. Over the course of the 120-min LC–MS run, 2340 MS-MS spectra were taken in CID mode, while 1896 ECD MS-MS spectra were recorded, a difference of only 25%. These data demonstrate not only that ECD data can be taken during an LC–MS run, but also that the amount of data generated almost matches traditional CID. 

Figure 3: Base peak ion chromatograms of the MS-MS spectra obtained during (a) LCâCID and  (b) LCâECD analyses of a human transferrin Lys-C digest. 

There are three N-linked glycosylation sites in human transferrin — Asn-432, 491, and 630 (5). We observed ions corresponding to the glycopeptides AA421-433 (3683 Da), AA490-508 (4465 Da), and AA619-646 (5500 Da) during a standard LC–MS experiment. However, we were unable to generate MS-MS spectra for these peptides using traditional CID. In the LC–ECD experiment, however, we were able to obtain ECD spectra for two of these glycopeptides, AA421-433 (

 1111.0, 4+) (Figures 4a and 4b, respectively). By combining the CID and ECD results, the sequence coverage was increased from 57% to 63%. More importantly, we were able to confirm the location of the glycosylation sites for these two peptides.  

Figure 4: ECD spectra of N-glycopeptides of transferrin: (a) AA421-433, 3683 Da, m/z = 921.5 (4+); (b) AA619-646, 5550 Da, m/z = 1111.0 (5+). 

Although the ability to perform ECD during an LC–MS run is advantageous, not all peptides have proven amenable to ECD fragmentation. One of the advantages provided by the QuECD platform is the ability to change the energy of the electron beam utilized in the fragmentation process. While traditional ECD reactions are performed at energies of < 1 eV, in HECD, the energy of the electron beam is increased to a value of 7–10 eV. Figure 5 shows ECD spectra of the AA619-646 peptide taken under regular ECD (Figure 5a) and HECD (Figure 5b) conditions. For both the c- and z-ion series, the number of observed ions is markedly increased under HECD conditions. While this ion was not identified using the traditional ECD data, it was easily identified using the richer HECD data.  

Figure 5: ECD spectra of transferrin's AA619-646 glycopeptide: (a) traditional ECD and (b) HECD. 

Modern biochemists require as many tools as possible in the effort to characterize protein structure and function. While MS has long been useful in these efforts, such experiments have proven costly and difficult. Advances in dissociation techniques have made progress in addressing these shortcomings. ECD is a powerful technique because of its utility in characterizing posttranslational modification as well as long peptides and small proteins. Because it can be implemented on a time-of-flight mass spectrometer, the QuECD platform has been successful in increasing the accessibility of ECD to nonspecialized laboratories. Further, QuECD allows for the analysis of complex mixtures via LC–ECD experiments. HECD increases the population of peptides that are amenable to ECD analysis. These advantages have brought QuECD to the forefront of structural protein characterization via MS. 

The authors would like to thank Takeshi Sakamoto of the Central Research Laboratory, Hitachi Ltd., for his help. 

 are with Hitachi High-Technologies America, Pleasanton, California.  

 are with Hitachi High-Technologies, Hitachinaka, Ibaraki, Japan.

(2) M.A. Shaw, M. Watanabe, T. Nabetani, Y. Hirabayashi, A. Tsuboyama, and C. Ostrander, 

(3) T. Baba, et al., Hitachi High-Technologies Application Note, NF/TN(E)-002, 2007. 

Articles

The authors discuss the use of electron-capture dissociation coupled with a linear ion trap time-of-flight mass spectrometer to investigate the structure of human transferrin.

Investigation of Transferrin Structure via Novel Electron Capture Dissociation Techniques Using a Hybrid Linear Ion Trap Time-of-Flight Mass Spectrometer

Successful characterization of protein posttranslational modifications (PTMs) is critical to our understanding of many biological processes. Unfortunately, attempts to describe PTMs often prove experimentally difficult and result in ambiguous conclusions. As technologies in the field of mass spectrometry (MS) continue to improve, people are turning increasingly to mass spectral techniques for PTM characterization. Recently, novel modes of peptide fragmentation have emerged that are giving scientists greater ability to elucidate protein posttranslational modification. One example is electron-capture dissociation (ECD), an alternative fragmentation mechanism for use in peptide analysis via tandem mass spectrometry. ECD selectively cleaves N-C(alpha) bonds of the peptide backbone, yielding c- and z-ions without the loss of labile PTMs. ECD therefore holds advantages over conventional fragmentation techniques such as collisionally induced dissociation (CID), which often cleave PTMs from the peptide backbone, precluding the ability to characterize them. Until recently, however, ECD was limited to resource-intensive Fourier transform–ion cyclotron resonance instruments. Here, we introduce a compact ECD device coupled to a linear ion trap  time-of-flight instrument, and use it to analyze protein phoshorylation in both offline (via direct infusion) and online (via nanoLC infusion) modes. We also utilize ECD to characterize both N- and O-linked peptide glycosylations. The results demonstrate ECD's capability to analyze a variety of posttranslational modifications using linear ion trap TOF-MS.

The majority of proteins are modified covalently in some manner prior to carrying out their role in the body. The nature of these modifications can vary significantly in both size and complexity, ranging from simple methylation and phosphorylation through more complex glycosylation and ultimately to small peptides and proteins such as ubiquitination and SUMOylation. Post-translational modifications (PTMs) are involved in a wide range of fundamental cellular processes, including enzyme regulation, signal cascade control, and cell–cell recognition and adhesion. PTMs are thereby likely involved in the mechanism of many disease classes, and are of great interest to the biochemical community. 

Traditionally, tandem mass spectrometry (MS-MS) has utilized collisionally induced dissociation (CID) to provide information on both protein sequence and post-translational modifications. CID-MS-MS produces b- and y-ions by activating the precursor ion collisionally with inert gas molecules. Unfortunately, information on the labile PTM often is lost during the CID process. For example, phosphorylated serine and threonine residues, which frequently are involved in a variety of intracellular signaling processes, have a tendency to lose their modifier phosphate groups via the β-elimination of a phosphoric acid molecule.  

In 1998, Zubarev and colleagues (1) reported electron-capture dissociation (ECD) as a new dissociation technique. ECD induces cleavage of peptide backbone bonds of multiply charged peptides and proteins via low energy (<1 eV) electron capture. While CID is induced by the excitation of vibrational energy in the analyte ions, ECD proceeds via intramolecular electron transfer (Figure 1). ECD randomly cleaves N-Cα bonds of the peptide backbone and yields c- and z-ions, regardless of their sequence, without the loss of labile modifications. ECD therefore allows both peptide identification and precise determination of the PTM location. 

Figure 1: A schematic illustration of ECD versus CID MS-MS. ECD-MS-MS cleaves the N-CÎ± bonds, and generates "c" and "z" fragment ions, while leaving the labile PTM intact.  

Although ECD has been recognized as a powerful tool for PTM analysis, traditionally it has required a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with an electrostatic magnetic field to provide low energy electrons. Such instruments are resource-intensive, require expert skill for operation, and are large and expensive. Moreover, ECD using FT-ICR MS requires long acquisition times because the intensities of the fragment ions are weak. For these reasons, the use of ECD has been limited in the field of basic research. 

Figure 2: A schematic diagram of the linear ion trap instrument equipped with a QuECD device between the linear ion trap, which isolates precursor ions and performs CID, and the quadrupole collision-induced dissociation (QCID) cell. 

In 2004, Baba and colleagues (2) succeeded in the achievement of ECD in a radio-frequency (RF) ion trap by using a linear ion trap device with a cylindrical permanent magnet. This small device, named QuECD, can be coupled to a linear ion trap TOF instrument to create a platform capable of high-throughput ECD (Figure 2) (2,3). Figure 3 shows the detailed structure of the ECD device. To avoid electron heating by the RF electric field in the ion trap, the electron beam is directed along the central axis of the linear ion trap. A magnetic field parallel to the central axis promotes electron travel along the central axis. The speed of the QuECD reaction is faster than conventional ECD using FT-ICR MS and is roughly equal to conventional CID. Because the reaction occurs on the chromatographic timescale, it can be performed during a standard liquid chromatography (LC)–MS experiment, which was unachievable previously with FT-ICR-based ECD techniques. 

Figure 3: A schematic diagram of the QuECD device. Low-energy electrons introduced from the top of the device are accelerated by a permanent magnet surrounding a radio-frequency linear ion trap. 

Here we use QuECD coupled with a linear ion trap TOF instrument (NanoFrontier eLD, Hitachi High Technologies America, Pleasanton, California) to analyze protein phosphorylation in both direct infusion and nanoLC modes. We also utilize this platform to characterize both N- and O-linked peptide glycosylations. The results demonstrate the capability of QuECD, in conjunction with linear ion trap TOF MS, to analyze a variety of PTMs. 

Conventional CID of phosphorylated peptides induces neutral loss of phosphoric acid, complicating determination of the peptide sequence and the location of phosphorylation. The analysis becomes increasingly difficult for multiply phosphorylated peptides. Figure 4 shows QuECD spectra of mono- and diphosphorylated versions of a peptide containing three tyrosine residues. The spectra indicate that Tyr12 of the monophosphorylated peptide and Tyr11 and Tyr12 of the diphosphorylated peptide are modified. These data demonstrate the utility of QuECD for determining the PTM locations of singly and multiply modified peptides. 

Figure 4: QuECD spectra of (a) mono- and (b) diphosphorylated peptides. Tyr12 in (a) and Tyr11 and Tyr12 in (b) are phosphorylated, respectively. 

As mentioned previously, the QuECD reaction occurs on the same timescale as conventional CID, which enables ECD to be performed during a standard LC–MS experiment. We used both LC–CID-MS–MS and LC–ECD-MS-MS to examine the phosphoproteome of an HEK293T cell lysate. Figure 5 compares sample MS-MS spectra of a triply charged phosphopeptide at 

 612.3 observed by both CID and ECD. In the case of CID-MS-MS, the loss of the phosphate group has impeded further fragmentation of the peptide. The resulting inadequate fragmentation pattern observed in the CID-MS-MS spectrum is in sharp contrast to the more complete fragmentation pattern observed via ECD-MS-MS. Using the ECD-MS-MS spectrum, this ion was identified as a peptide from an insulin-like growth factor II receptor that contains a phosphorylation at Serine 2484. 

Figure 5: Comparison of typical CID- and QuECD-MS-MS spectra encountered in the phosphoproteome study of an HEK293T cell lysate. Both spectra are product ion spectra of the precursor ion [M + 3H]3+ at m/z 612.3 that correspond to a peptide spanning residues 2477 to 2491 of the human protein IGF2R. The newly identified phosphoserine at position 2484 is denoted in red type. 

We found that the combination of CID and ECD data together provides the most comprehensive analysis of phosphorylated peptides. The peak lists obtained by CID- and ECD-MS-MS experiments were merged, and the resulting composite data set was searched against the Swiss-Prot database using the MASCOT search engine. More phosphorylated proteins and peptides were identified using the combined data set than those from individual searches. These results demonstrate that ECD is an effective technique complementary to CID and that a combination of the two dissociation modes provides an excellent platform for phosphoproteome analysis. 

To identify a glycopeptide, the following three factors must be determined: the amino acid sequence of the peptide, the location of glycosylation, and the carbohydrate structure. ECD simultaneously identifies an amino acid sequence and glycosylation sites by fragmenting peptides while leaving the carbohydrate chain intact. The ECD spectrum in Figure 6a shows data generated from an N-linked glycopeptide. It suggests that the interval between C3 and C4 ions exactly corresponds to the sum of the molecular weight of N-glycan coupled with asparagine. The amino acid sequence and binding site (N) are both determined without dissociating the carbohydrate chain. Figure 6b illustrates the analysis of an O-linked glycopeptide with three possible glycosylation sites (S). The ECD spectra suggest that the interval between z7 and z6 (or between c5 and c6) ions exactly corresponds to the sum of the molecular weights of the O-glycan coupled with a serine, uniquely determining the glycan binding site. Again, the amino acid sequence is clearly determined from c- and z-type fragment ions without dissociating the carbohydrate chain. 

Figure 6: QuECD spectra of (a) N-linked and (b) O-linked glycopeptides. 

The results indicate that QuECD, coupled with the linear ion trap TOF MS, creates an efficient tool for analysis of PTMs such as phosphorylation and glycosylation. The results also show that the combination of CID and ECD data result in improved reliability and depth of phosphoproteome analysis. Because the QuECD reaction occurs on the chromatographic timescale, this platform is capable of running LC–ECD-MS-MS experiments, allowing for ECD interrogation of complex samples.  

The authors would like to thank Naomi Manri of the Central Research Laboratory, Hitachi Ltd., for his help. 

 is with Hitachi High-Technologies, Hitachinaka, Ibaraki, 312-8504, Japan. 

 are with Brain Science Institute, RIKEN, Wako, Saitama, 351-0198, Japan. 

The authors introduce a compact ECD device coupled to a linear ion trap time-of-flight instrument, and use it to analyze protein phosphorylation in both offline and online modes.

Akira Tsuboyama

Articles

Investigation of Transferrin Structure via Novel Electron Capture Dissociation Techniques Using a Hybrid Linear Ion Trap Time-of-Flight Mass Spectrometer

Posttranslational Modification Characterization via Electron Capture Dissociation Using a Linear Ion Trap Time-of-Flight Mass Spectrometer