Micro‐high‐performance liquid chromatography/Fourier transform mass spectrometry with electron‐capture dissociation for the analysis of protein enzymatic digests

Davidson, Walter; Frego, Lee

doi:10.1002/rcm.666

Cited by 32 publications

(26 citation statements)

References 21 publications

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“…The current ECD protocol remains tedious and time-consuming. The low dutycycle has imposed additional constraints on the implementation of the ECD technique for use in on-line liquid chromatography MS/MS analysis [17,18]. To improve the duty-cycle, Zubarev and co-workers have replaced the heated filament with an indirectly heated dispenser cathode [19].…”

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confidence: 99%

Optimization of experimental parameters for electron capture dissociation of peptides in a Fourier transform mass spectrometer

Chan

2002

J. Am. Soc. Mass Spectrom.

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This paper describes our effort in optimizing the experimental parameters for electron capture dissociation (ECD) of peptides in a commercially available Fourier-transform mass spectrometer. Using a built-in electrically heated filament electron gun, it was demonstrated that good quality ECD spectra of peptides (MW Ͻ 2500) could be obtained by irradiating the isolated peptide molecule-ions with a short pulse (50 ms) of low-energy (3-6 eV) electrons. In addition, we have also demonstrated that pulsing of inert cooling gas (argon) could further improve the intensity of the ECD-induced fragment ions. Due presumably to the influence of the strong magnetic field on the trajectories of electrons, the distance between the electron gun and the trapped-ion cell (i.e., 108 mm versus 20 mm) was found to have little influence on the efficiency of the ECD process(es). From a systematic study on the impact of the filament heating current, filament bias voltage, and electron irradiation time on the intensities of precursor ions and various fragment ions, it was postulated that subsequent capture of electrons by the fragment ions, i.e., neutralization of the fragment ions, might be a significant event for limiting the intensity of the fragment ions. . For peptide analysis, all these methods result mainly in cleavage of the peptide amide bonds, leading to the N-terminal b-type and C-terminal y-type ions [6]. Since the implanted energy is normally randomized in various vibrational degrees of freedoms prior to fragmentation, bond cleavage tends to occur primarily at the weakest linkages in the peptides, such as on the N-terminal side of a proline residue or on the C-terminal side of aspartic acid [7]. As a consequence, complete peptide sequence is rarely obtained from a tandem mass spectrum using the above ion activation method.Electron Capture Dissociation (ECD) was first introduced [8] by McLafferty and co-workers in 1998 and is a relatively new mass spectrometry technique for inducing dissociation of multiply-charged ions. By irradiating the multiply-charged peptide/protein ions with low-energy (Յ0.2 eV) electrons, the peptide/protein ions would undergo exothermic capture of electrons and thus leading to the formation of a reduced radical ion, [M ϩ nH] (nϪ1)⅐ϩ , which rapidly dissociates via backbone N™C cleavage to form predominantly series of c-type and z⅐-type ions. As a result of the non-ergodic nature of ECD process [8], strong backbone bonds can also be cleaved even in the presence of much weaker bonds [9]. It has been demonstrated that this dissociation technique offers more extensive and nonspecific fragmentation, resulting in more extensive sequence information. For instance, collision-induced dissociation of multiply-charged ubiquitin, 8.6 kDa, resulted in only 25% of sequence specific information, whereas electron capture dissociation on collisional-activated ubiquitin ions could lead to complete sequence information [10].Although it has previously been shown that this dissociation technique can provide important s...

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confidence: 99%

Optimization of experimental parameters for electron capture dissociation of peptides in a Fourier transform mass spectrometer

Chan

2002

J. Am. Soc. Mass Spectrom.

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“…The problem was addressed by the introduction of the heated dispenser cathode by Tsybin in 2001 [113]. The timescale for ECD was reduced significantly (ms) and it became possible to couple ECD with online liquid chromatography [114][115][116]. Palmblad et al [115] coupled LC with ECD for the analysis of a tryptic digest of bovine serum albumin and Davidson and Frego [114] demonstrated LC-ECD analysis of a pepsin digest of cytochrome c. Zubarev and co-workers demonstrated an approach on a modified LTQ FT hybrid linear ion trap FTICR in which a low resolution survey scan in the ion trap was followed by analysis of CID and ECD in the ICR cell [117].…”

Section: Ie Formation Of [M+(n-1)h]mentioning

confidence: 99%

“…The timescale for ECD was reduced significantly (ms) and it became possible to couple ECD with online liquid chromatography [114][115][116]. Palmblad et al [115] coupled LC with ECD for the analysis of a tryptic digest of bovine serum albumin and Davidson and Frego [114] demonstrated LC-ECD analysis of a pepsin digest of cytochrome c. Zubarev and co-workers demonstrated an approach on a modified LTQ FT hybrid linear ion trap FTICR in which a low resolution survey scan in the ion trap was followed by analysis of CID and ECD in the ICR cell [117]. Creese and Cooper [118] undertook a direct comparison of LC-ECD-MS/MS with LC-CID-MS/MS revealed that, CID resulted in greater overall protein coverage, whereas ECD gave higher confidence in peptide sequence, thus showing the complementary nature of CID and ECD for bottom-up proteomics.…”

Section: Ie Formation Of [M+(n-1)h]mentioning

confidence: 99%

Top-down proteomics: applications, recent developments and perspectives

Pamreddy

Panyala

2016

J Appl Bioanal

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REVIEWNow-a-days, top-down proteomics (TDP) is a booming approach for the analysis of intact proteins and it is attaining significant interest in the field of protein biology. The term has emerged as an alternative to the well-established, bottom-up strategies for analysis of peptide fragments derived from either enzymatically or chemically digestion of intact proteins. TDP is applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein complexes and assemblies. This article delivers an overview of the methodologies in top-down mass spectrometry, mass spectrometry instrumentation and an extensive review of applications covering the venomics, biomedical research, protein biology including the analysis of protein post-translational modifications (PTMs), protein biophysics, and protein complexes. In addition, limitations of top-down proteomics, challenges and future directions of TDP are also discussed.

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“…As a consequence, the electron irradiation time required exceeded several seconds and tens of seconds, with some exceptions. 28,58 The novel electron-injection systems based on indirectly heated cathodes provided increased electron flux, larger overlap of ion and electron clouds and better control of the electron energy. As a result, the reliability of ECD was improved and the reaction rate was increased by about two orders of magnitude.…”

Section: Electron Capture Dissociation Ratementioning

confidence: 99%

Peptide and protein characterization by high‐rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

et al. 2004

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The analytical utility of the electron capture dissociation (ECD) technique, developed by McLafferty and co-workers, has substantially improved peptide and protein characterization using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The limitations of the first ECD implementations on commercial instruments were eliminated by the employment of low-energy electron-injection systems based on indirectly heated dispenser cathodes. In particular, the ECD rate and reliability were greatly increased, enabling the combination of ECD/FTICR-MS with on-line liquid separation techniques. Further technique development allowed the combination of two rapid fragmentation techniques, high-rate ECD and infrared multiphoton dissociation (IRMPD), in a single experimental configuration. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation and led to improved peptide and protein characterization. The application of high-rate ECD/FTICR-MS has demonstrated its power and unique capabilities in top-down sequencing of peptides and proteins, including characterization of post-translational modifications, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation). Analysis of peptide mixtures has been accomplished using high-rate ECD in bottom-up mass spectrometry based on mixture separation by liquid chromatography and capillary electrophoresis. This paper summarizes the current impact of high-rate ECD/FTICR-MS for top-down and bottom-up mass spectrometry of peptides and proteins.

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Micro‐high‐performance liquid chromatography/Fourier transform mass spectrometry with electron‐capture dissociation for the analysis of protein enzymatic digests

Cited by 32 publications

References 21 publications

Optimization of experimental parameters for electron capture dissociation of peptides in a Fourier transform mass spectrometer

Optimization of experimental parameters for electron capture dissociation of peptides in a Fourier transform mass spectrometer

Top-down proteomics: applications, recent developments and perspectives

Peptide and protein characterization by high‐rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

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