In-line purification of equilibrium mixtures Melissa Graewert June - - PowerPoint PPT Presentation

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in line purification of equilibrium mixtures
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In-line purification of equilibrium mixtures Melissa Graewert June - - PowerPoint PPT Presentation

Sep 09, 2022 144 likes •633 views

In-line purification of equilibrium mixtures Melissa Graewert June 25 th The odd one out Polydisperse samples Aggregates intrinsic property of the sample eg. monomer-dimer-oligomer equilibrium or incomplete formation of

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In-line purification of equilibrium mixtures

Melissa Graewert June 25th

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The odd one out

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Polydisperse samples

  • Aggregates
  • intrinsic property of the sample
  • eg. monomer-dimer-oligomer equilibrium
  • r incomplete formation of complexes
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Native Gel Electrophoresis Size Exclusion Chromatography Dynamic Light Scattering Static Light Scattering

PREPARING THE EXPERIMENT

Analytical ultracentrifugation

percentage m/z)

nativeMS

  • Sample Characterization
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  • Size Exclusion

Chromatography

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  • J. Synchrotron Rad. (2004)
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25/06/2016 8

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.

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The SAXS instrument at the Barkla Laboratory of

  • Biophysics. The set-up

includes a Dectris PILATUS 300K-20Hz detector, three pin- hole optics and Rigaku FR-E+ Superbright X- ray generator.

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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, automation RALS  MW UV  concentration (when dn/dc not known (lipids, DNA, etc.))

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

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Light absorbance: ~ c, ε

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Light absorbance: ~ c, ε Refraction: ~ c, dn/dc dual cell, deflection design

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Light absorbance: ~ c, ε Refraction: ~ c, dn/dc dual cell, deflection design

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Light absorbance: ~ c, ε Scattering: ~ c, dn/dc, MW Refraction: ~ c, dn/dc

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  • 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

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

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Step 1: identification of buffer region (RI signal)

  • Automated M.O.S.E.S.
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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.
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Step 1: identification of buffer region (RI signal) Step 2: Buffer subtraction, I(0) and RG determination of each curve Step 3: Correlation of the SAXS data with MALVERN data Step 4: Extraction of biophysical information for each frame

  • Automated M.O.S.E.S.
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Step 1: identification of buffer region (RI signal) Step 2: Buffer subtraction, I(0) and RG determination of each frame Step 3: Correlation of the SAXS data with MALVERN data Step 4: Extraction of biophysical information for each frame

  • Automated M.O.S.E.S.
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Step 1: identification of buffer region (RI signal) Step 2: Buffer subtraction, I(0) and RG determination of each frame Step 3: Correlation of the SAXS data with MALVERN data Step 4: Extraction of biophysical information for each frame Step 5: Determination of “peak region”, scaling and averaging of respective curves Step 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.
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  • Automated M.O.S.E.S.
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  • 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

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

— PlaB (batch, 4.5mg/ml) — PlaB, tetrameric peak lg I(q), a.u. q, nm-1

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  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

— PlaB (batch, 4.5mg/ml) — PlaB, tetrameric peak lg I(q), a.u. q, nm-1

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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 lg I(q), a.u. q, nm-1

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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 lg I(q), a.u. q, nm-1

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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 lg I(q), a.u. q, nm-1

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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

lg I(q), a.u. q, nm-1

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  • Simultaneous Data Collection
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Electron micrograph of Legionella pneumophila wwww.wikimedia.org

  • M.O.S.E.S. (Microsplitting for Online Separation,

Extended characterization and SAXS analysis)

host pathogen Lipolytic active monomeric PlaB Activation Via dimeric state Self protection through inactive tetrameric PlaB

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  • 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)
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  • M.O.S.E.S. 2.0
  • HPLC pumps
  • Autosampler
  • Even more automation
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Native Gel Electrophoresis Size Exclusion Chromatography Dynamic Light Scattering Static Light Scattering

PREPARING THE EXPERIMENT

Analytical ultracentrifugation

percentage m/z)

nativeMS

  • Sample Characterization
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Katja Kuhle Antje Flieger Robert Koch Institute THE SAXS GROUP

Special Thanks!!!