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In-line purification of equilibrium mixtures Melissa Graewert June 25 th
In-line purification of equilibrium mixtures
Melissa GraewertJune 25th
The odd one out
Polydisperse samples
• Aggregates• intrinsic property of the sample
• eg. monomer-dimer-oligomer equilibrium • or incomplete formation of complexes
Native Gel Electrophoresis
Size ExclusionChromatography
Dynamic Light Scattering
Static Light Scattering
PREPARING THE EXPERIMENT
Analytical ultracentrifugation
perc
enta
ge
m/z)
nativeMS
Sample Characterization
o Size ExclusionChromatography
J. Synchrotron Rad. (2004)
25/06/20168
Information extracted from the elution profile of an initially polydisperse solution of commercial BSA. Column: SHODEX 402.5-4F. Flow rate: 150 µl min−1. Injected volume: 5 µl at 88.8 g l−1. (a) Chromatogram of the elution profile. The complex profile indicates the presence of several species. The main peak, at 17.04 min, corresponds to BSA monomer.
The SAXS instrument at the Barkla Laboratory of Biophysics. The set-up includes a DectrisPILATUS 300K-20Hz detector, three pin-hole optics and RigakuFR-E+ Superbright X-ray generator.
SEC – SAXS but some disadvantages:
Analysis of thousands successive scattering curves a posteriori
No knowledge of the solute concentration Uncertainity about equilibrium driven systems further characterisation useful for automation and
validation Add MALVERN BOX
RI concentration, automationRALS MW UV concentration (when dn/dc not known (lipids, DNA, etc.))
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
Light absorbance: ~ c, ε
Light absorbance: ~ c, ε
Refraction: ~ c, dn/dc
dual cell, deflection design
Light absorbance: ~ c, ε
Refraction: ~ c, dn/dc
dual cell, deflection design
Light absorbance: ~ c, ε
Scattering: ~ c, dn/dc, MW
Refraction: ~ c, dn/dc
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
Independent Molecular Weight determination
Concentration of single frames (Scaleing, MW)Automation
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
Step 1: identification of buffer region (RI signal) Automated M.O.S.E.S.
Step 1: identification of buffer region (RI signal)Step 2: Buffer subtraction, I(0) and RG determination of each curve
Automated M.O.S.E.S.
Step 1: identification of buffer region (RI signal)Step 2: Buffer subtraction, I(0) and RG determination of each curveStep 3: Correlation of the SAXS data with MALVERN dataStep 4: Extraction of biophysical information for each frame
Automated M.O.S.E.S.
Step 1: identification of buffer region (RI signal)Step 2: Buffer subtraction, I(0) and RG determination of each frameStep 3: Correlation of the SAXS data with MALVERN dataStep 4: Extraction of biophysical information for each frame
Automated M.O.S.E.S.
Step 1: identification of buffer region (RI signal)Step 2: Buffer subtraction, I(0) and RG determination of each frameStep 3: Correlation of the SAXS data with MALVERN dataStep 4: Extraction of biophysical information for each frameStep 5: Determination of “peak region”, scaling and averaging of respective curvesStep 6: Handing over of final curve to down-stream analysis
MW expected
MW SLS
MW Porod
MW Modell
MW I(0)
45 kD 48 kD 50 kD 46 kD 39 kD
Automated M.O.S.E.S.
Automated M.O.S.E.S.
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
Phospholipase B of Legionella pneumophila (Lpn PlaB)
Kuhle et al.; J Biol Chem. 2014 Jul Gräwert et al.; Scientific Reports online June 2015
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
— PlaB (batch, 4.5mg/ml)— PlaB, tetrameric peak
lgI(q
), a.
u.
q, nm-1
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
— PlaB (batch, 4.5mg/ml)— PlaB, tetrameric peak
lgI(q
), a.
u.
q, nm-1
MWRALS = 230±15 kD
MWI(0 )= 225±15 kD
MWVol = 170±30 kD
MWDAMMIF= 203±30 kD
MWSEC ~ 100 kD
— PlaB (batch, 4.5mg/ml)— PlaB, tetrameric peak
lgI(q
), a.
u.
q, nm-1
MWRALS = 230±15 kD
MWI(0 )= 225±15 kD
MWVol = 170±30 kD
MWDAMMIF= 203±30 kD
MWSEC ~ 100 kD
— PlaB (batch, 4.5mg/ml)— PlaB, tetrameric peak
lgI(q
), a.
u.
q, nm-1
MWRALS = 230±15 kD
MWI(0 )= 225±15 kD
MWVol = 170±30 kD
MWDAMMIF= 203±30 kD
MWSEC ~ 100 kD
— PlaB (batch, 4.5mg/ml)— PlaB, tetrameric peak
lgI(q
), a.
u.
q, nm-1
MWRALS = 230±15 kD
MWI(0 )= 225±15 kD
MWVol = 170±30 kD
MWDAMMIF= 203±30 kD
MWSEC ~ 100 kD
— PlaB (batch, 4.5mg/ml)— PlaB, tetrameric peak--- PlaB, dimeric peak
lgI(q
), a.
u.
q, nm-1
Simultaneous Data Collection
Electron micrograph of Legionella pneumophila wwww.wikimedia.org
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
hostpathogen
Lipolytic activemonomericPlaB
ActivationVia dimericstate
Self protectionthrough inactivetetrameric PlaB
M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)
• Alternative strategy to study (moderatly) polydisperse samples• Required sample amounts: at least 50 ul of > 5mg/ml• Sufficient buffer• Optimize your SEC run• If possible collect batch sample as well• Check for radiation damage, add 3% glycerol (if feasible)
M.O.S.E.S. 2.0
HPLC pumps Autosampler Even more automation
Native Gel Electrophoresis
Size ExclusionChromatography
Dynamic Light Scattering
Static Light Scattering
PREPARING THE EXPERIMENT
Analytical ultracentrifugation
perc
enta
ge
m/z)
nativeMS
Sample Characterization
Katja Kuhle Antje FliegerRobert Koch Institute
THE SAXS GROUP
Special Thanks!!!
