See the linked Application Note for a demonstration of phosphopeptide analysis using e-MSion’s ExD Cell in an Agilent 6550 iFunnel Q-TOF.
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 – 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.
Top-Down Protein Sequencing
Figure 1 – 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.
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.
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.
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