EXAMPLE DATA

ECD Fragmentation for the Workhorse Mass Spectrometer

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.

Aspartate/Isoaspartate Characterization

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)].

 

 

Leucine/Isoleucine Differentiation

e-MSION 6545XT delight

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.

 

Phosphorylation Site Localization

Figure 1 – ECD product ion mass spectrum of the [M+3H]3+ CK1 phosphopeptide precursor, acquired using the ExD Cell in an Agilent 6460 triple quadrupole. Inset: magnified region showing evidence of phosphorylation at the seventh residue in the form of the the c72+ fragment ion.

 

Figure 2 – Top: ECD product ion mass spectrum of the [M+20H]20+ precursor of the serine phosphorylated protein alpha-casein, acquired using the ExD Cell in a Thermo Fisher Scientific Orbitrap Q Exactive. Sequence coverage was calculated by ProSight Lite to be 35%, using centroids with S:N > 2 and mass error < 10 ppm. Single-residue-resolution evidence confirmed the presence of two out of the eight phosphoserine residues. Middle & Bottom: Magnified regions showing evidence of phosphorylation at the 46th and 48th residues, respectively.

 

Figure 3 – ECD product ion mass spectrum of the serine phosphorylated, lysine acetylated Biotin-labeled Histone H3 peptide (residues 1-21), acquired using the ExD Cell in a Thermo Fisher Scientific Orbitrap Q Exactive. The mass range 1700-3000 has been magnified 25X to emphasize the z15-z17 fragment ions indicating the presence of phosphorylated serine.

 

Top-Down Protein Sequencing

Figure 1 – Left: ECD product ion mass spectrum of the quasi-native [M+6H]6+ precursor of Ubiquitin, acquired using the ExD Cell in a Bruker ultrOTOF-Q. Right: Sequence coverage map generated by LCMS Spectator. After manual curation, sequence coverage by c and z type ions was determined to be 100%, excluding proline residues.

Figure 2 – Top: Averaged ECD product ion mass spectrum of the denatured [M+20H]20+ precursor of bovine carbonic anhydrase, acquired using the ExD Cell in an Agilent 6545XT Q-TOF. Twenty five product peaks were manually assigned. Bottom: Sequence coverage map generated by ProSight Lite from peaks with S:N greater than 10 and mass error less than 10 ppm.  Sequence coverage by b, y, c, and z type ions was reported to be 64%, although some of the manual assignments were missed by ProSight.

Uncongested Spectra

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.

 

Compatibility with HPLC, UPLC, IMS, CE

Figure 1 – The ExD Cell operates at speeds greater than the fastest separation technique, and has been successfully tested with HPLC, IMS, and CE.

 

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.

 

Single-Residue-Resolution HX-MS

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.

 

Disulfide Bond Localization without Prior Reduction

See Fort et al. J Proteome Res 17:926-933 (2018) for data regarding selective disulfide dissociation.

Visit our Publications page to view more published and presented data.