In-line purification of equilibrium mixtures. Melissa Graewert June 25 th

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1 In-line purification of equilibrium mixtures Melissa Graewert June 25 th

2 The odd one out

3 Polydisperse samples Aggregates intrinsic property of the sample eg. monomer-dimer-oligomer equilibrium or incomplete formation of complexes

4 Sample Characterization Analytical ultracentrifugation nativems percentage Native Gel Electrophoresis m/z) Size Exclusion Chromatography Dynamic Light Scattering Static Light Scattering PREPARING THE EXPERIMENT

5 o Size Exclusion Chromatography

6 J. Synchrotron Rad. (2004)

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8 Information extracted from the elution profile of an initially polydisperse solution of commercial BSA. Column: SHODEX F. 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 min, corresponds to BSA monomer. 8 25/06/2016

9 The SAXS instrument at the Barkla Laboratory of Biophysics. The set-up includes a Dectris PILATUS 300K-20Hz detector, three pinhole optics and Rigaku FR-E+ Superbright X- ray generator.

10 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.))

11 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)

12 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)

13 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)

14 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)

15 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)

16 Light absorbance: ~ c, ε

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

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

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

20 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

21 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)

22 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)

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25 Automated M.O.S.E.S. Step 1: identification of buffer region (RI signal)

26 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

27 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 Step 3: Correlation of the SAXS data with MALVERN data Step 4: Extraction of biophysical information for each frame

28 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 frame Step 3: Correlation of the SAXS data with MALVERN data Step 4: Extraction of biophysical information for each frame

29 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 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

30 Automated M.O.S.E.S.

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32 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 Jul Gräwert et al.; Scientific Reports online June 2015

33 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis)

34 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis) lg I(q), a.u. PlaB (batch, 4.5mg/ml) PlaB, tetrameric peak q, nm -1

35 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis) lg I(q), a.u. PlaB (batch, 4.5mg/ml) PlaB, tetrameric peak q, nm -1

36 lg I(q), a.u. PlaB (batch, 4.5mg/ml) PlaB, tetrameric peak MW RALS = 230±15 kd MW I(0 ) = 225±15 kd MW Vol = 170±30 kd MW DAMMIF = 203±30 kd MW SEC ~ 100 kd q, nm -1

37 lg I(q), a.u. PlaB (batch, 4.5mg/ml) PlaB, tetrameric peak MW RALS = 230±15 kd MW I(0 ) = 225±15 kd MW Vol = 170±30 kd MW DAMMIF = 203±30 kd MW SEC ~ 100 kd q, nm -1

38 lg I(q), a.u. PlaB (batch, 4.5mg/ml) PlaB, tetrameric peak MW RALS = 230±15 kd MW I(0 ) = 225±15 kd MW Vol = 170±30 kd MW DAMMIF = 203±30 kd MW SEC ~ 100 kd q, nm -1

39 lg I(q), a.u. PlaB (batch, 4.5mg/ml) PlaB, tetrameric peak --- PlaB, dimeric peak MW RALS = 230±15 kd MW I(0 ) = 225±15 kd MW Vol = 170±30 kd MW DAMMIF = 203±30 kd MW SEC ~ 100 kd q, nm -1

40 Simultaneous Data Collection

41 M.O.S.E.S. (Microsplitting for Online Separation, Extended characterization and SAXS analysis) Lipolytic active monomeric PlaB Activation Via dimeric state host pathogen Electron micrograph of Legionella pneumophila wwww.wikimedia.org Self protection through inactive tetrameric PlaB

42 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)

43 M.O.S.E.S. 2.0 HPLC pumps Autosampler Even more automation

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45 Sample Characterization Analytical ultracentrifugation nativems percentage Native Gel Electrophoresis m/z) Size Exclusion Chromatography Dynamic Light Scattering Static Light Scattering PREPARING THE EXPERIMENT

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47 Katja Kuhle Antje Flieger Robert Koch Institute Special Thanks!!! THE SAXS GROUP