CH908, Problem set 9. Electron Capture Dissociation (ECD) of peptides
1. Calculate the ECD fragments that would be expected from the following
peptide sequence: RGHTTPSDELWIVK. What can you say about expected
relative cleavage intensity? Create a table of the expected fragments and mark
them with "strong, medium, weak" for expected intensities.
Relative fragmentation efficiency will be pretty even, with the exception of a lack
of cleavage at proline - because it's ECD - and low abundance at glycine.
Because the n-terminus has arginine, and the c-terminus has lysine, the
neutralized proton will be primarily the one on the c-terminal lysine, so nterminal fragments will dominate. Thus you'll see mostly c-ions, with only a few
z-ions.
Also, because of the many hydrogen bonds in this system (many -OH groups
and acids to bond with the charge sites), the fragment intensity for a 2+ ion will
be low unless a bit of heating (activated ion ECD) is used to break the hydrogen
bonds - and even then, formation of c• and z (even electron) fragment ions will
be readily observable.
There's a reported, slight tendency for higher cleavage at tryptophan, but it's
barely noticeable compared to the proline effect in CAD.
2. Calculate the ECD fragments that would be expected from the following
peptide sequence: RGHTpTPpSDELpYIVK. Note: pS, pT, and pY refer to
phosphoserine, phosphothreonine, and phosphotyrosine, respectively. What
can you say about expected relative cleavage intensity? Create a table of the
expected fragments and mark them with "strong, medium, weak" for expected
intensities.
Same fragments as above. However, the phosphates will primarily remain intact
on the peptide rather than being cleaved as they would in CAD.
There will be a strong charge state dependence of fragmentation in this case
because of tendency of phosphate groups to be negatively charged. It will also
often be difficult to observe significant positive ion signal for this species for the
same reason.
3. ECD can be used to distinguish leucine and isoleucine. How? Draw a
probable radical rearrangement that could distinguish these two. Under what
conditions?
Isoleucine:
Leucine:
This will occur under slightly more energetic conditions than normal ECD, with
electrons in the 10 eV range, rather than the usual <1 eV range. This
experiment is known as "hot" ECD or HECD.
4. In the following paper, a novel hemoglobin variant was discovered using
electron capture dissociation. Which one? How?
J. P. Williams, A. J. Creese, D. R. Roper, B. N. Green, H. J. Cooper, Hot
Electron Capture Dissociation Distinguishes Leucine from Isoleucine in a Novel
Hemoglobin Variant, Hb Askew, beta 54(D5)Val -> Ile. J. Am. Soc. Mass
Spectrom. 2009, 20. 1707-1713, DOI: 10.1016/j.jasms.2009.05.002.
Doh! The answer is in the title. Valine54 to Isoleucine or V54I.
It was detected using ECD because it's the only current method that can
distinguish leucine and isoleucine.
5. In the following paper, variants of Amyloid Beta (Aβ) were generated that
contained a mixture of Aspartic acid and IsoAspartic acid at particular residues.
How were they generated? Which residues? How was this tested? Were the
results conclusive?
N. P. Sargaeva, C. Lin, P. B. O'Connor, Identification of Aspartic and Isoaspartic
Acid Residues in Amyloid beta Peptides, Including A beta 1-42, Using ElectronIon Reactions. Anal. Chem. 2009, 81. 9778-9786, DOI: 10.1021/ac901677t.
Note: these papers is also posted on the CH908 website on the recommended
texts section.
Doh! Another mistake, the variants were aspartic acid and isoaspartic acid, not
leucine and isoleucine.
Ms. Sargaeva used a couple of commercially available variants, D7N and D23N
which are known to correlate with increased risk of Alzheimer's disease. Aging
these samples converted the asparagines back to aspartic acid, but as a mixture
of two isoforms, normal aspartic acid (Asp), and beta-linked aspartic acid (called
isoaspartic acid, isoAsp).
ECD clearly distinguishes Asp and isoAsp because of cleavage at the cα-cβ
bond, which results in two diagnostic fragments, c+57 and z-57. They are
definitely conclusive, although one was a bit less convincing from the intensities.