Masaki Watanabe is with Hitachi High-Technologies, Hitachinaka, Ibaraki, Japan.

Masaki Watanabe

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.

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

Mass spectrometers are effective for identifying and quantifying unknown molecules, such as disease-related proteins and small molecules in pharmaceutical research and medical diagnosis. In addition, mass spectrometry (MS) can be particularly powerful when analyzing molecules with complex structures, such as posttranslationally modified proteins. Among various MS approaches, high-resolution multistep tandem MS (MS-MS) is an emerging methodology for accurate identification of complex molecules. In this article, we describe a new approach for mass analysis with enhanced quantitative capability combined with high-resolution multistep MS-MS, where the dynamic range of quantitation covers four orders of magnitude.

Mass spectrometry (MS) is used widely to identify and quantify unknown molecules in a broad range of applications, such as pharmaceutical development and medical diagnosis. To clarify disease mechanisms, for example, MS is used to search for disease-related molecules, or biomarker molecules, such as proteins and metabolites. For such study of biomarker molecules, both qualitative and quantitative approaches are important; qualitative analysis discovers and identifies the structure of biomarker molecules, and quantitative analysis typically compares the amount of biomarkers in disease-state and healthy-state samples. Research on currently unidentified or unknown molecules relevant to specific biological and chemical functions of importance would be most effective when these qualitative and quantitative approaches are consolidated. 

For the analysis of unknown molecules, ion traps and time-of-flight (TOF) mass analyzers are used widely because of their high speed in acquiring full mass spectra, thus facilitating discovery and identification of new biomarker candidates. Although ion trap analyzers have moderate mass accuracy, mass resolution, and quantitative dynamic range, they are unique in their ability for multistep MS-MS (MS

), which is advantageous in extracting molecular structure information on small molecules and posttranslational modified proteins (1–4). TOF mass-analyzers, on the other hand, are superior in mass accuracy, mass resolution, and quantitative dynamic range. Compared to ion traps, TOF analyzers offer higher mass accuracy and a wider dynamic range, which is advantageous for accurate determination of elemental composition of molecules. 

An emerging new approach in the mass analysis of unknown molecules is a combination of the best of these two mass analyzers: high-resolution multistep MS-MS (5–14), which is a hybrid of an ion trap and a TOF mass analyzer. This hybrid system offers high mass resolution, quantitative dynamic range, and MS

Figure 1 shows schematically the structure of the high-resolution multistep tandem mass spectrometer (NanoFrontier LD, Hitachi High Technologies America, San Jose, California), which is a liquid chromatography (LC)–MS system in which the mass spectrometer is a hybrid linear-ion-trap (LIT) TOF mass analyzer (14).  

To optimize the TOF analyzer design for increased dynamic range, the TOF analyzer is coupled in an orthogonal arrangement relative to the LIT, so that the TOF response is highly linear to the sample concentration over a wide range. An important device for this orthogonal arrangement is a collisional-focusing chamber placed between the LIT and the TOF, in which the ion beam is focused into the TOF by a quadrupole ion-guide under collision with buffer gas. This region also can be used as a dissociation chamber. 

  mode, ions that are created in the ion source are transferred to the TOF analyzer through LIT. In the MS

  modes, the precursor ions are first isolated in the LIT and dissociated either in the LIT or in the collisional-focusing chamber. 

In addition to the TOF analyzer arrangement, data-acquisition electronics that convert the ion signal to a mass spectrum are important in obtaining a wide dynamic range. For example, response saturation during data acquisition can result in decreased dynamic range performance. Conventional time-to-digital conversion (TDC) data-acquisition electronics are prone to this saturation because TDC has a dead time after an ion arrival at the detector. During this dead time, TDC cannot detect subsequent ion arrival, resulting in saturation during high ion intensity. To eliminate this saturation limit by TDC, the MS system is equipped with 2-GHz analog-to-digital conversion (ADC) data-acquisition electronics, which convert full temporal train of detector signal to digital without a conversion dead time. The result is a higher resistance to detector saturation and improved dynamic range compared with TDC. 

The LC system for the MS system can either be a semimicro flow LC or a nanoflow LC (nanoLC) system, depending upon application. The nanoflow LC system is uniquely suited to the quantitative analysis of proteins because of its accurate reproducibility in chromatographic retention time for trace samples. It is capable of gradient flow at 50–250 nL/min rates with high stability and high reproducibility even at 50 nL/min, which is achieved by a splitless gradient mechanism called dual exchange gradient system (DEGS) (19,20).  

Figure 2a shows a typical quantitative dynamic range of the mass spectrometer for reserpine. It shows a linear dynamic range of more than four orders of magnitude, spanning from 10 fg to 200 pg, for which the orthogonal arrangement of the TOF analyzer and the ADC data-acquisition electronics contribute considerably.  

Figure 2b shows a typical mass spectrum of insulin, where the resolving power for quadruple charged ions of insulin is over 10,000, which is adequate for most high-resolution applications. 

Therefore, the high-resolution multistep tandem mass spectrometer has high resolution combined with a wide dynamic range, which is suitable for accurate qualitative and quantitative analysis of unknown molecules and biomarkers. 

Two Dissociation Modes in the LIT and in the Collision Chamber

For best performance for a broad range of application, two distinct modes of collisionally induced-dissociation (CID) are available with the MS system, which is realized by its unique orthogonal arrangement of LIT and TOF: LIT-CID and quadrupole-CID (or Q-CID). In the LIT-CID mode, ions are dissociated inside the LIT with capability for multistep MS-MS analyses (MS

), while in the quadrupole-CID (or Q-CID) mode, ions are dissociated inside the collisional-focusing chamber.  

  in a LIT is a useful tool for the analysis of complex molecular structures, it is limited by a low-mass cutoff, resulting in difficulty detecting low mass fragment ions of 

 less than ¼ or ⅓ of the precursor ion. On the other hand, MS-MS analysis with the quadrupole-CID is characterized by the production of fragment ions of lower mass compared with that produced using LIT.  

Figure 3 shows a comparison of the two types of MS-MS fragmentation modes, LIT and Q-CID, for a peptide [Glu]1-fibrinopeptide B. In the Q-CID MS-MS mode, ions with lower mass were observed, but they were not observed using the LIT MS-MS mode. The lower-mass ions consist of b2 ions, produced via fragmentation of sample peptide at the second peptide bond from the N-terminus, and y1 ions, produced via fragmentation at the peptide bond of the C-terminus. Thus, the MS-MS spectrum obtained using quadrupole CID yielded more information regarding amino acid sequence than that obtained via LIT. The method also might be beneficial for the structural analysis of small molecules, such as metabolites, where one might need information of low-mass fragment ions.  

  measurement with LIT and MS-MS measurement with a quadrupole collision chamber, to support a variety of samples and purposes. 

Software for the Efficient Search of Biomarker Molecules: Information-Based Acquisition 

  capability efficiently, it is important that the mass spectrometer can control the spectral acquisition sequence intelligently.  

The software for this purpose in the MS system is called information-based acquisition (IBA) (21,22) and has two major characteristic functions: real-time construction of an exclusion database and real-time intelligent triggering of MS

In the real-time construction capability of exclusion database, which is shown schematically in Figure 4a, IBA can store 

 and retention time information of the precursors, which have been analyzed already via MS-MS into an internal database in real time. During repeated LC–MS measurements of the same sample, subsequent measurements can reference the internal database and avoid duplication of the MS-MS spectra of identical precursor ions. By employing the IBA capability, as more measurements are repeated, an increased number of less abundant proteins–peptides, for example, can be identified. The IBA software allows editing of the internal database as well. Known information regarding interfering molecules, as shown in Figure 4a, can be registered in advance.  

  upon user-designated conditions on the MS

  spectrum. For example, in protein applications, IBA can analyze de-novo sequence of the precursor peptide from the MS

  fragment peaks (21) in real time. If information in MS

  is not sufficient for de-novo sequencing of more than 

  to obtain further fragmentation information. The 

 value is designated by the user. IBA can also trigger MS

  when a user-designated neutral-loss peak is found in the MS

Figure 4b schematically depicts one procedure of using IBA for search of biomarkers. First, Sample A is measured to register information regarding the tandem-analyzed target precursors into the database in realtime. Next, during measurements of Samples B and C, the database is used as a reference to selectively obtain the MS-MS spectrum of a target component that is not included in Sample A. Reference 23 describes an application of such an IBA procedure for analysis of an unknown biomarker protein in serum. 

A combination of high-resolution and multistep tandem analysis is advantageous for structure determination of complex molecules. Figure 5 shows an example of high-resolution LC–MS

  analysis of a sample containing a family of erythromycins (erythromycins A to F) in which the major component was erythromycin A. Erythromycins B–F initially were unknown impurity components identified by this high-resolution LC–MS

Figure 5a shows the structure and fragmentation diagram of erythromycin A and its MS

  spectrum corresponds to a substructure composed of the amino sugar (denoted by substructure b) and the macrolide ring (substructure c), where the neutral sugar (substructure a) was removed as a neutral loss. The major mass peak 

  spectrum corresponds to the amino sugar, where the macrolide ring was removed from the MS

The base peak ion chromatogram is shown in Figure 5b where five impurity peaks are observed together with the major erythromycin peak, which was more than 140 times as intense as the smallest impurity peak, peak 1. The five impurity peaks were subjected to high-resolution MS

  analysis similar to Figure 5a. Summary of the experiment is shown in the table of Figure 5c. The elemental composition can be deduced from the high accuracy 

  spectrum for each impurity peak, which were within 5 ppm error from theoretical 

 values. Similarly, the elemental compositions for the substructures of the neutral sugar substructure (a), amino sugar (b), and the macrolide ring (c) all can be deduced from the high-resolution MS

  results shown in Figure 5c, the impurity peaks were identified to erythromycins B–F with high confidence, as indicated in Figure 5d. Thanks to the wide dynamic range of the methodology, high-resolution MS

  analysis with a good signal-to-noise ratio was possible, which resulted in high-confidence identification even for the most minor impurity peak, whose intensity was less than 0.7% of the major component. 

An Example of Nonlabeled Relative Quantitative Analysis of Proteins

In protein research, nonlabeled relative quantitation of a mixture of proteins between different samples often is used to search for biomarkers. Because mass chromatographic peaks must be compared between samples, reproducibility of the retention times of the peaks is important. The highly reproducible nanoflow LC portion of the LC–MS system is ideally suited for this application. To evaluate accuracy in such a relative quantitation experiment, two artificially prepared peptide mixture samples were analyzed as follows. 

Both peptide mixture samples, samples A and B, were tryptic digests of four kinds of proteins: yeast enolase, phosphorylase b, yeast alcohol dehydrogenase, and bovine hemoglobin. The concentrations of phosphorylase b, yeast alcohol dehydrogenase, and bovine hemoglobin before digestion were 100 nmol/L in both samples, while the concentration of yeast enolase prior to digestion was 100 nmol/L for sample A and 200 nmol/L for sample B. A 1-μL volume of each sample was injected into the nanoLC system and detected by MS, resulting in MS spectra of the peptide mixture and MS-MS fragment mass spectra of peptides. 

Relative-quantitative performance is shown in Figure 6. Figure 6a shows the total ion chromatograms of samples A and B. Here, the peptide peaks are well resolved with highly reproducible retention times. The chromatographic peaks, which correspond to the peptides, are categorized into two groups: peaks whose intensities are nearly equal for samples A and B, and peaks whose intensities are twice greater for sample B compared with sample A. Twice-greater peaks in sample B are derived from tryptic-digest peptides of yeast enolase, which is consistent with the sample preparation. Thus, these total ion chromatograms suggest a quantitative performance and qualitative selectivity suitable for relative-quantitation by LC–MS.  

To characterize the performance more quantitatively, the proteins were quantified relatively as follows. First, to identify the proteins from the peptide fragment spectra, the MS-MS spectra were analyzed with the protein search software MASCOT (Matrix Science, Inc., Boston, Massachusetts), which resulted in the reliable assignment of each peptide peak to one of the four proteins. Next, intensity of each peptide mass-chromatographic peak was calculated relative to a major peptide peak of the bovine hemoglobin. Then, the ratio of the corresponding peak intensity in sample B to sample A was calculated. After grouping the identified peptide peaks according to their parent proteins, the relative peak-intensity ratios of the peptides belonging to a same protein were averaged to give an average relative-intensity-ratio between samples A and B for that protein, which is shown in Figure 6b.  

For phosphorylase b and yeast alcohol dehydrogenase, the ratio of sample B to sample A was 1.16 ± 0.13 and 1.19 ± 0.16, respectively, while the ratio of yeast enolase in sample B to sample A was 1.95 ± 0.05. Thus, the relative quantitation experiment was accurate within 16% of the actual amounts of proteins in the sample, where the experiment includes not only LC–MS-MS, but also protein pretreatment such as digestion.  

Improved identification and quantification abilities using high-resolution multistep MS-MS are expected to accelerate the qualitative discovery and quantitative validation analysis of unknown candidate molecules of chemical and biological relevance, such as biomarker molecules critical to medical diagnosis and pharmaceutical development. 

Izumi Waki, Katsuhiro Kanda, Izumi Ogata, Tsukasa Shishika, Akira Owada, Shinji Yoshioka, Masaki Watanabe, 

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

is with Hitachi High Technologies America, San Jose, California. 

Masaki Watanabe

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