Presentation by: Mahati Mokkarala
Date of Presentation: 12/4/12
Paper 1: Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T. Ion mobility-mass
spectrometry reveals a conformational conversion from random assembly to b- sheet
in amyloid fibril formation. Nat. Chem. 3, 172-177 (2010)
Paper 2: Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers, M.T. Ab(39-42)
Modulates Ab Oligomerization but not Fibril Formation. Biochemistry. 51, 108-177
(2011)
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Effective method for determining compound
chemical structure, protein modification
patterns, interactions, etc.
Protein Sample/
(can be liquid, other
states)
Ionization- Hard or
Soft methods
(conversion to
gaseous state) Ex:
for proteins, ESI
(nano), MALDI with
lasers
Mass Spectrometer
machine- mass
analyzer/detector.
Example: Time of
Flight Mass
Spectrometer
(TOF), Quadrupole
Mass Spectrometer
(QMS)
-Detects m/z z/n of
various ion
fragments
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Ion mobility devices separate (peptide
sequence) ions based on particle mobility,
shape, charge
Easily pair ion mobility with mass spectra and
ionization devices [5]
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• Peptide ion fragments enter chamber filled with
gas (buffer gas, chiral selectivity element, etc)
• Ion mobility delayed- ‘friction’ collisions with gas
molecules- propelled by electric field (Image from source [2])
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Linear Drift Time (LDT) Mass Spectrometry‘easier’ calculation correlation between
collision cross section and drift time for ions
Traveling Wave Ion Guide (TWIG) IM-MS
Field Asymmetric Ion Mobility Mass
Spectrometry (FAIM) [4].
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LDT gas tube- with weak
electric field- constant drift
velocity
 Can average collisions to get
the collision cross section
 Advantages: high resolution,
easier to quantify degree of
ion separation
 Disadvantages: low ‘drift
cycle’ need to constantly
introduce a pulse of ions- can
promote wasting of a large
portion of sample source [4]

Image from source [4] (see works cited), in
source reprinted by permission from source [20]
in paper
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(K naught) Reduced mobility ~ 1/W (W =
Collision Cross section Image from source [4]
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How to calculate K naught? K simplified,
related to drift time P- pressure, V- voltage,
linear relationship Image from source [2]
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Bowers et al. Paper 2, summarizes key
relationship between s (collision crosssection) and drift time
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q = ion charge, T = temperature, m = reduced
mass, N = He/gas number density, l = drift cell
length
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Paper 1: Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T.
Ion mobility-mass spectrometry reveals a conformational
conversion from random assembly to b- sheet in amyloid fibril
formation. Nat. Chem. 3, 172-177 (2010)
9
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Detection oligomer shifts- tough to
characterize due to quick conformational
shifts
With IM-MS, could greater determine at
oligomer combination (n) globular-b sheet
transformation occurs.
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•amyloid-forming yeast prion
protein Sup35 (NNQQNY)
•human insulin regions- (VEALYL)
•human islet amyloid
polypeptide- (SSTNVG)
•YGGFL- usually forms an
exclusively isotropic not fibril
structure
Peptides exposed to
following apparatus:
And
then
ESI/Quadrople Mass Spec.
Image from:
http://chemwiki.ucdavis.edu
IM (from source [3]
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IM (time of delay) – calculate s for each
oligomer (size n)
 Compare collision cross section per oligomer
number (n) with theoretical s(n) for
fibril/isotropic growth
Isotropic Growth formula:
Fibril Growth formula:
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Mass Spectra- indicates oligomerization due to large n/z observed for
two peptides-YGGFL, VEALYL (one isotropic growth control, other
fibril
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Shows sample ATD intensity captures by IM-MS for the NNQQNY peptide;
broad peaks- correlate to multiple oligomer combination states- use average
drift time for calculations
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Can clearly
correlate
experimental
collision cross
section per
each oligomer
combination
with calculated
theoretical s(n)
Top: for YGGFL
Second: for
NNQQNY
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Experimental data
and proposed
oligomerization for
peptide VEALYL (c
)
and peptide
SSTNVG (d )
Indicates peptide
(c ) –initiates with
single strand fibril
before at n =5
switching to the
zipper form
Peptide (d)isotropic until n =
12/14, consists of
both zipper and
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Verification of fibril formation at specified oligomer (n) verified by
AFM visualization of each protein mixture sample
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With IMS-MS, now cab follow through
peptide self-assembly step by step from an
oligomer of 1 for given peptide fragment
Stresses importance of the IMS-MS
technique can learn more on at what state
oligomer-b fibril transformation occurs
Very relevant for greater study of amyloid b
caused diseases
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Paper 2: Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers,
M.T. Ab(39-42) Modulates Ab Oligomerization but not Fibril
Formation. Biochemistry. 51, 108-177 (2011)
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Mechanism of Ab(39-42) binding to Ab42 or
Ab40 tough to experimentally verify via X ray
crystollagraphy or NMR
IM-MS and molecular dynamic simulations as
well as ThT assays- further verify Ab(39-42)
interactions with Ab42 and Ab40
Why important?: CTF Ab(39-42) known to
inhibit Ab toxicity
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IM- (nanospray) ESI- quadrople mass
spectra- oligomer disassociation due to
Ab(39-42) (CTF)
ThT fluorescence assay- does Ab(3942)influence/limit fibril formation?
Modeling software- AMBER force field
simulation, SHAKE- verify possible
binding/structure Ab(39-42) with Ab peptide
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Results- Definite
difference in mass spectra
peaks between both
spectra a- Amyloid
particle alone, b- Amyloid
plus 1:5 CTF added
-Key peaks to focus on in
b figure: z/n = -5/2 peak –
one CTF/dimer
z/n = -3, 1 or 2 CTF bound
to single oligomer (Ab42)
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n/z = -5/2 m/z = ~ 1800 for dimer
peak- does CTF prevent
dodecamers? Ans: Yes.
Prevention of
dodecamers,
decamers
requires high
(1:5)
concentration
CTF
n/z = -5/2 for Ab42 particle –
Does CTF reverse Ab
aggregation? Ans: Yes
Incubation of
select amyloid
dimer peaks for
2 hours prior to
exposure to
CTF
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Question: Does CTF bind to
tetramers, hexamers, dimers
of amyloid b42? Ans: Yes
Expose m/z = 1884 peak with
bound CTF to dimers to IMMS indicate definite cross
sections for dimer, tetramer,
hexamer
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Similar experiment
repeated for the
Ab40 peptide- as
above, observe
distinctive peaks z/n
= -4, -3 for one or
two CTF- binding to
single oligomer
IM-MS indicatesno shift oligomer
size with CTF,
same as peptide
CTF- interacts
with Ab40
One dimer-CTF
species identified
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Both CTF plus Ab40/Ab42
peptides with MTT assayPC12 cells promotes cell
viability –importance of
breaking toxic oligomer
aggregates
ThT fluorescence- EM microscope
visualization
-Fluorescence increase-fibrils
--Oligomers eventually to fibrils even with CTF
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Observe structure if CTF binds to
Ab more than 20 times, adds to
being in a bound state, etc
Calculate cross sections of
structures (long collision integral)
Compare structures to Mass Spec
experimental data
Observe: CTF fragments bind: N, C
terminus, internal regions via van
der Walls interactions of Ab42
peptide.
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With IMS-MS techniques:.
 CTF binds (Van der Waals) with monomeric, 2,4,6 Amyloid b 42
particles
 CTF disassociates dodecamers into non toxic oligomers
 Ab40 – binds with two CTF via electrostatic interaction, no
disaggregation oligomers
 CTF binding- C, N terminus, internal structures Amyloid b 42
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[1] Bleiholder C., Dupuis N F., Wyttenbach T. & Bowers, M.T. Ion mobility-mass spectrometry reveals a
conformational conversion from random assembly to b- sheet in amyloid fibril formation. Nat. Chem. 3,
172-177 (2010)
[2] “Theories and Analysis.” The Bower’s Group UC Santa Barbara.
<http://bowers.chem.ucsb.edu/theory_analysis/> . Accessed: December 3, 2012.
[3] Gessel M.M., Wu C., Li H., Nitan G., Shea J. & Bowers, M.T. Ab(39-42) Modulates Ab Oligomerization
but not Fibril Formation. Biochemistry. 51, 108-177 (2011)
[4] Harvey S.R. MacPhee C.E., Barran P.E. Ion mobility mass spectrometry for peptide analysis. Methods.
54(4), 454-461 (2011)
[5] Kanu A.B., Dwivedi P., Tam M., Matz L., Hill H.H., Ion mobility- mass spectrometry. Journal of Mass
Spectrometry. 43, 1-22 (2008)
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