Methods Native and HDX Mass Spectrometry HydrogenDeuterium exchange Engen et al Weis Int Rev Phys Chem 2013 HDX MS Experimental setup D 2 O Quench ID: 931784
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Slide1
Project Z1:'Biophysical Methods'
Native and H/DX Mass Spectrometry
Hydrogen/Deuterium
exchange
Engen et al., Weis,
Int
Rev Phys Chem (2013)
H/D-X MS: Experimental setup
D
2
O
Quench
reaction
at various time points
Digest
Intact protein
UPLC separation
ESI-TOF MS
Data
analysis
Deuterium incorporation curves
Variation of reaction mixture/conditions
Automated system is necessary to enable reproducibility !
ESI-TOF MS
UPLC
separation
Quench reaction
at various time points
Digest
ACQUITY UPLC M-class HDX with automation
Native MS of non-covalent protein complexes
:
Historical Example: Analysis of the
sHsps
TaHsp16.9
Complex stoichiometry
Sobott
et al., Robinson, J.
Biol
Chem
(2002)
Dynamics of the complex: Subunit exchange
Example
: Analysis
of
Src-kinase mutations
Boczek et al., Buchner
,
PNAS (2015)
Peptide 162-172: SH2-domain
no
significant differences:Mutation does not change dynamics of this region
significant differences:Mutation changes dynamics of this region
Peptide 270-278: b1-strand & P-loop
Allosteric changes in the dynamics
Colors: increased H/DX
increased dynamicsData provide restraints for MD simulations
point mutation
Native MS
H/DX MS
MS
Experiments & setup
Service measurements
Use of instruments
D
ata analysis
& evaluation
SPRannual training course
SAXSannual training course
Native & H/DX MS
annual training course monthly discussion group
Training courses
User support
Z1
Structures
Baumeister
: A01
Groll: A02, Conti: A07
NMR
Sattler: B03,
Hagn
: B13
Mass spectrometry
Sieber A09, Hartl A12
Organic chemistryLang: B10
Biophysical chemistryBuchner: A03, A06Schwenkert: A04
Further collaborations: Itzen: B05, Kapurniotu B06, Reif B07
Cell biology
Feige: B11
Support
Ralf
Strehle
Martin HaslbeckGina FeindPhD students from project A12 and A03
People dedicated