Transcript Lecture 13

CH 908: Mass Spectrometry
Lecture 13
Non-covalent interactions, protein
conformations, and protein
complexes
Prof. Peter B. O’Connor
Objectives
• Proteins can retain some structure when transitioning
from solution phase to gas phase.
• This results in:
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Non-covalent interaction studies
protein higher-order structural studies
protein complex studies
Ligand-binding studies
• Methodologies
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ESI tricks
Hydrogen/Deuterium Exchange (HDX)
Crosslinking
Tagging/labeling
ECD
Leucine Zipper Data
50 μM
N16V supposed to
dimerize more strongly
than GCN4-p1
Li, Y. T.; Hsieh, Y. L.; Henion, J. D.; Ganem, B.; Senko, M. W.; McLafferty, F. W. Mass Spectrometric Studies on Noncovalent Dimers of Leucine
Zipper Peptides J. Am. Chem. Soc. 1993, 115, 8409.
Pitfalls in the use of charge states
for conformational determination
• Stability. Protein conformations are unstable under most
MS experimental conditions. Extreme care must be
taken and controls must be run to be able to say
anything definitive
• Is the conformational change you are detecting related in
any way to solution phase conformation? How?
Carol Robinson’s research group
at Cambridge (Now Oxford)
H/D exchange
• Solution phase
– Walter Englander (non-MS, biochemist; Pennsylvania)
http://hx2.med.upenn.edu/
– measurement of unfolding rates (D. Smith, Nebraska)
– AA positional measurement of unfolding rates using NSCAD-FTMS (I. Kaltashov, Massachusets)
– Julia Chamot-Rooke / Guillaume v.d. Rest (Paris)
• Gas phase
– Cyt. C has different conformations in the gas phase (F.
Mclafferty)
– Ubiquitin/Cyt. C/KIX folding/unfolding in the gas phase
probed by ECD
H/D Exchange of CRABP
1.Eyles, S. J.; Dresch, T.; Gierasch, L. M.; Kaltashov, I. A. Unfolding dynamics of a beta-sheet protein studied by mass spectrometry J. Mass
Spectrom. 1999, 34, 1289-1295.
MS/MS of CRABP during H/D back-exchange
1.Eyles, S. J.; Dresch, T.; Gierasch, L. M.; Kaltashov, I. A. Unfolding dynamics of a beta-sheet protein studied by mass spectrometry J. Mass
Spectrom. 1999, 34, 1289-1295.
Melting Curves
PLIMSTEX: Simple titration using deuterium incorporation
as the readout
Gas phase HD exchange
1.Wood, T. D.; Chorush, R. A.; Wampler, F. M. I.; Little, D. P.; O'Connor, P. B.; McLafferty, F. W. Gas Phase Folding and Unfolding of Cytochrome c
Cations Proc. Nat. Acad. Sci. USA 1995, 92, 2451.
Gas phase HD exchange
1.Wood, T. D.; Chorush, R. A.; Wampler, F. M. I.; Little, D. P.; O'Connor, P. B.; McLafferty, F. W. Gas Phase Folding and Unfolding of Cytochrome c
Cations Proc. Nat. Acad. Sci. USA 1995, 92, 2451.
Pitfalls to H/D exchange experiments
• Nothing is more hygroscopic than water
(the backexchange problem)
• How much is water involved in protein
folding?
• When performing MS/MS experiments on
H/D exchanged proteins, is there proton
scrambling?
Chemical Labeling of surface
groups
• Tolan collaboration
• oxidation of surface groups
• ICAT reagent
1.Hopkins, C. E.; O'Connor, P. B.; Allen, K. N.; Costello, C. E.; Tolan, D. R. Chemical-modification rescue assessed by mass
spectrometry demonstrates that gamma-thia-lysine yields the same activity as lysine in aldolase Protein Science 2002, 11, 1591-1599.
UV releases ·OH from
H2O2 to oxidize
surface residues on
proteins
Sharp, J. S.; Becker, J. M.; Hettich, R.
L. Analysis of protein solvent
accessible surfaces by photochemical
oxidation and mass spectrometry Anal.
Chem. 2004, 76, 672-683.
Cys > Trp, Tyr > Met > Phe >
His > Ile > Leu > Pro
Top-down localization of modifications
Sharp, J. S.; Becker, J. M.; Hettich, R. L. Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry Anal. Chem.
2004, 76, 672-683.
ICAT Method
Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb,
M. H.; Aebersold, R., Quantitative analysis of
complex protein mixtures using isotope-coded
affinity tags Nature Biotechnology 1999, 17, 994999.
Pitfalls to chemical labeling
• Unexpected reactions
– non specific reagents
– reactions at other amino acids
• Complex spectra – how do you fish out the
peak that has been modified?
– Metal containing tags…
– isotopically labeled tags…
Crosslinking
• Mann
• Pierce catalog
• biotinylated crosslinker
Crosslinking
Method
Crosslinking proteins
Kruppa, G. H.; Schoeniger, J.; Young, M. M. A top down approach to protein structural studies using chemical cross-linking and Fourier transform
mass spectrometry Rapid Commun. Mass Spectrom. 2003, 17, 155-162.
Ubiquitin Crosslinked
Kruppa, G. H.; Schoeniger, J.; Young, M. M. A top down approach to protein structural studies using chemical cross-linking and Fourier transform
mass spectrometry Rapid Commun. Mass Spectrom. 2003, 17, 155-162.
Pitfalls to crosslinking experiments
• heterogeneity of the crosslinking sites
– very complex spectra very quickly
• non-specificity of the crosslinking
chemistry
• cross-reactions
• zerolength crosslinkers are sometimes
useful
Ion Mobility
Spectrometry
• indirectly
measures crosssectional area
Purves, R. W.; Barnett, D. A.; Guevremont, R.,
Separation of protein conformers using electrospray-high
field asymmetric waveform ion mobility spectrometrymass spectrometry Int. J. Mass Spectrom. Ion Processes
2000, 197, 163-177.
IMS-TOF
http://www.youtube.com/watch?v=tGO2nZq-q5E
Pitfalls of Ion Mobility Spectrometry
• Gas phase structure – does it mean
anything?
• Accelerating through the IMS could unfold
the ions
Use of ECD for
protein folding
studies
Breuker, K.; Oh, H.; Horn, D. M.; Cerda, B. A.; McLafferty, F. W. Detailed unfolding and folding of gaseous ubiquitin ions characterized by electron
capture dissociation J. Am. Chem. Soc. 2002, 124, 6407-6420.
Refolding of Gas phase proteins
probed by ECD
Breuker, K.; Oh, H.; Horn, D. M.; Cerda, B. A.; McLafferty, F. W. Detailed unfolding and folding of gaseous ubiquitin ions characterized by electron
capture dissociation J. Am. Chem. Soc. 2002, 124, 6407-6420.
Proposed Ubiquitin
Gas phase structures
– probed by
photofragmentation
Oh, H.; Breuker, K.; Sze, S. K.; Ge, Y.; Carpenter, B. K.; McLafferty, F. W.
Secondary and tertiary structures of gaseous protein ions characterized by
electron capture dissociation mass spectrometry and photofragment spectroscopy
Proc. Nat. Acad. Sci. USA 2002, 99, 15863-15868.
Native ECD
Breuker, K.; McLafferty, F. W. Native electron capture dissociation for the structural characterization of noncovalent interactions in native cytochrome
c Angewandte Chemie-International Edition 2003, 42, 4900-4904.
Fundamental points about
conformational studies
• While exciting, these are often very difficult
experiments, requiring a lot of time and good
control experiments
• The data one gets is often ambiguous, but no
more so than solid state structures obtained in
X-Ray crystallography
• It’s crucial to differentiate between solution state
and gas phase conformations – they are very
different
Direct ESI-MS of non-covalent
complexes
• Ganem/Henion
• Robinson
• Loo
Leucine Zipper Data
50 μM
N16V supposed to
dimerize more strongly
than GCN4-p1
Li, Y. T.; Hsieh, Y. L.; Henion, J. D.; Ganem, B.; Senko, M. W.; McLafferty, F. W. Mass Spectrometric Studies on Noncovalent Dimers of Leucine
Zipper Peptides J. Am. Chem. Soc. 1993, 115, 8409.
Transthyretin
Sobott, F.; McCammon, M. G.; Robinson, C. V. Gas-phase dissociation pathways of a
tetrameric protein complex Int. J. Mass Spectrom. Ion Processes 2003, 230, 193-200.
Pitfalls of non-covalent studies
• Adequate controls MUST be run
• Concentration dependence MUST be
established
• Solution conditions are crucial
• Ionization conditions are crucial
• Removing solvent MAY change
conformation and cause dissociation of
a weakly bound species
Screening
• Griffey/Hofstadler's PNA-RNA work
• Hunt's MHC peptides
RNA’s with a neutral
mass tag
Hofstadler, S. A.; Sannes-Lowery, K. A.; Crooke, S. T.; Ecker, D. J.; Sasmor, H.; Manalili, S.; Griffey, R. H. Multiplexed screening of neutral masstagged RNA targets against ligand libraries with electrospray ionization FTICR MS: A paradigm for high-throughout affinity screening Anal. Chem.
1999, 71, 3436-3440.
RNA Screening
Hofstadler, S. A.; Sannes-Lowery, K. A.; Crooke, S. T.; Ecker, D. J.; Sasmor, H.; Manalili, S.; Griffey, R. H. Multiplexed screening of neutral masstagged RNA targets against ligand libraries with electrospray ionization FTICR MS: A paradigm for high-throughout affinity screening Anal. Chem.
1999, 71, 3436-3440.
Antigen presentation
Denhaan, J. M. M.; Meadows, L. M.; Wang, W.; Pool, J.; Blokland, E.; Bishop, T. L.; Reinhardus, C.; Shabanowitz, J.; Offringa, R.; Hunt, D. F.;
Engelhard, V. H.; Goulmy, E. The Minor Histocompatibility Antigen Ha-1 - a Diallelic Gene With a Single Amino Acid Polymorphism Science 1998,
279, 1054-1057.
Pitfalls to High Throughput
screening using ESI-MS
• Appropriate controls MUST be run to
verify regularly that non-covalent
binding is working.
• Solution/Source conditions are crucial.
• Reproducibility of the method is crucial.
• ESI-MS screening experiments bias
against weakly bound species.
Fundamental points about noncovalent adducts
• Some non-covalent adducts are very
strongly bound and can be stronger than
weak covalent bonds
• It’s important to distinguish between
specific and non-specific adduction
Sometimes, noncovalent interactions are stronger than
covalent bonds!
Self Assessment Questions
• With ESI, protein conformations are
somewhat preserved upon entering the
gas phase. Why is this significant?
• What can non-covalent interaction studies
tell us about proteins?
• How can HDX be used to study protein
structure using a mass spectrometer?
Would CAD be useful here? ECD?
CH908: Mass spectrometry
Lecture 1
Fini…
Methods:
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Analysis of Charge State Distributions
H/D exchange
Labeling
Crosslinking
MS/MS
Ion Mobility Spectrometry
Electron Capture Dissociation
Analysis of Charge State
Distributions
• Loo/Smith
• Cassady
• Kaltashov