ECD Fragmentation for the Workhorse Mass Spectrometer
Example Data

The ExD cell produces ECD fragmentation similar to FTICR-ECD, but on workhorse mass spectrometers like Q-TOFs and Orbitrap QEs. The ability to perform simple electron-based fragmentation enables valuable new types of data and time-saving workflows. The Example Data on this page illustrates the value the ExD cell can add to your laboratory.

Click the toggles below to see detailed figures and captions describing some ExD example data.

Example Data

Figure 1 – The ExD Cell can produce side-chain fragmentation useful for distinguishing leucine from isoleucine residues. Isoleucine loses 29 Da to form a w ion, while leucine loses 43 to form a w ion. The peptide ECDD(isoD)ELIGHTFLK was run on an Agilent 6545XT Q-TOF equipped with the ExD Cell. Top: The diagnostic loss of 43 Da from the z7 fragment indicates the presence of leucine at position 7. Bottom: The diagnostic loss of 29 Da from the z8 fragment indicates the presence of isoleucine at position 8.

ECD is so delightful!

Figure 1 – ECD product ion mass spectrum of the [M+2H]2+ peptide precursor ECDD[isoD]ELIGHTFLK, acquired using the ExD Cell in an Agilent 6545XT Q-TOF. The 57 Da loss from z10+, corresponding to the loss of C2HO2, is diagnostic for isoaspartate [Cournoyer et al. Protein Sci 14(2):452-63 (2005)].

 

Figure 1 – ECD product ion mass spectrum of the [M+6H]6+ precursor of Ubiquitin. Product ions were mainly c and z type, minimizing peak overlap while still yielding excellent sequence coverage.

Figure 1 – ECD product ion mass spectrum of phosphorylated Tyrosine Kinase Peptide 3, acquired using the ExD cell in an Agilent 6545XT Q-TOF. Peak assignments made using LCMSSpectator with S:N > 3 and mass error < 5 ppm.


Figure 2 – ECD product ion mass spectrum of a phosphorylated, acetylated, and biotin-labeled Histone H3 peptide (1-21), acquired using the ExD cell in an Agilent 6545XT Q-TOF. Peak assignments made using LCMSSpectator with S:N > 3 and mass error < 5 ppm.

Click here to download the Application Note for a demonstration of phosphopeptide analysis using e-MSion’s ExD Cell in an Agilent 6550 iFunnel Q-TOF.

“The widespread availability, low cost, and robustness of ion trap instruments as well as its compatibility with chromatographic time scales largely helped to popularize ETD, making it nowadays the preferred ExD technique in the field of bottom-up proteomics. Notably, a compact ECD cell that can be implemented on Orbitrap and Q-TOF platforms may rejuvenate ECD in the near future.” (Potel et. al. 2019)

Figure 1 – The ExD Cell enables single-residue-resolution HX-MS without hydrogen scrambling. Top Left: ECD product ion mass spectrum of the [M+3H]3+ precursor of the HX-MS model peptide “P1”, acquired using the ExD Cell in an Agilent 6545 Q-TOF. The His-rich N-terminal half of P1 is engineered to exchange hydrogen more quickly than its C-terminal half. Following back-exchange of deuterated P1, N-terminal c fragments are expected to retain relatively little deuterium, while C-terminal c fragments are expected to retain more deuterium. Any deviations from this pattern would be indicative of hydrogen migration, or “scrambling”, as a result of vibrational excitation. Top Right: After allowing P1 to back-exchange for 5 minutes, deuterium content by residue was similar to expected results published by Rand et al. JACS 130: 1341 (2008): N-terminal residues exchanged hydrogen more quickly than C-terminal residues. Bottom Left: The isotopic distribution of back-exchanged (purple) c5 fragment is similar to protonated (black). Bottom Right: The isotopic distribution of back-exchanged (purple) c9 fragment is shifted right relative to protonated (black), indicating residual deuterium incorporation.

Figure 2 – Top: HPLC chromatogram of cyanogen bromide-digested hemoglobin. Bottom: ECD product ion spectrum of hemoglobin 1-32 peptide.

Figure 3 – Top: Ion Mobility drift spectrum revealing two conformers of [M+3H]3+ Substance P. Bottom: The ExD Cell was used to generate ECD product ion mass spectra of the two different Substance [M+3H]3+ conformers. Subtle differences in the types and amounts of ECD fragments were observed. Data courtesy of Cathy Costello, Boston University.

Figure 4 – Top: Capillary Electropherogram of a 4-protein test mixture containing intact lysozyme, ubiquitin, carbonic anhydrase, and alpha-synuclein. Middle: ECD product ion mass spectrum of alpha-synuclein averaged over 3 scans, acquired using the ExD Cell in an Agilent 6550 Q-TOF. Bottom: Alpha-synuclein sequence coverage by ECD product ions from these 3 scans was calculated by LCMS Spectator to be 90%. Data courtesy of Michael Knierman, Eli Lilly.

Figure 1 – Insulin structure and fragments identified in an LC/ECD experiment (average of 5 scans). c– and z-type ECD ions are indicated with blue dots, green dots indicate b– and y-type ions, and purple dots signify a-type ions. Circled dots indicate w-ions. Top: fragments found with Cys7-Cys7 disulfide bridge assumed to be intact. Each chain was calculated separately. The fragments of the B-chain were calculated with the mass of the A-chain as a modification on Cys7, and fragments of the A-chain were calculated with the mass of the B-chain on its Cys7. Bottom: fragments found with the Cys19-Cys20 disulfide bridge intact. The fragments were calculated in the same way as the upper figure, with the mass of the A-chain on Cys19 of the B-chain and the mass of the B-chain on Cys20 of the A-chain.

Figure 2 – Insulin structure and fragments identified by CID, 25 V collision energy.

Slicing the Gordian Knot: Protein Disulfide Mapping with ExD | Application Note / English / August 2021 / 2.8 MB / PDF