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Expanding Surface Plasmon Resonance Capabilities with Reichert
- 1. Paving the Way for New Applications through Flexible Biomolecular Interaction Analysis
- 2. Reichert Life Science Legacy • 100+ year optics history – Instruments with leading sensitivity, robustness and efficiency • 20 years of SPR expertise • >250 publications on Reichert SPR • Cover the full spectrum of bio-molecular interactions – Protein-protein, Protein-DNA, Protein-carbohydrate, etc. – Protein-small molecule – Whole cells, viruses • Ensuring success through superior support & service – SPR support staff helps researchers solve problems – Methods development, high-volume experiments, feasibility studies A history of exceptional performance, value and support.
- 3. Reichert SPR Product Offerings • Innovative two and four channel systems – High sensitivity for low molecular weight analysis & increased confidence • Noise and sensitivity performance required for challenging applications • More applications and sample types – Robust enough for handling crude samples, cell lysates, aggregates • High sample capacity – Two 96- or 384-well plates – Up to 768 samples—or any combination of plates and vials • Scalable to meet your needs now and later – Solutions grow as you do – Professional services available
- 4. What is Surface Plasmon Resonance? Metal Surface Plasmons (Electron Waves) Surface Plasmon Resonance Angle Intensity > θc Resonance (Energy Transfer)
- 6. Response Units Time Response Units are in µRIU (10-6 refractive index units) 1 µRIU = 1 pg/mm2 of mass binding A very precise refractometer A very precise mass sensor
- 7. Versatile Technique Time Response Binding Reponse Baseline Is there an interaction? (Yes/No Binding) How strong is the interaction? (Affinity) How quickly do they interact and dissociate? (Kinetics) Why? (Thermodynamics) (∆H, ∆S, ∆G) How much? (Concentration) Regeneration
- 8. All Classes of Biomolecules <100 Da to Proteins to Cells • Proteins • Lipids • Carbohydrates • Nucleic Acids • LMW Molecules • Whole Cells • Bacteria, Viruses
- 9. Low Volume Flow Cell Sensor Slide Inlet from Injector Outlet from Left Channel Outlet From Right Channel or Both Channels 2CH 4CH
- 10. Reichert Sensor Surfaces (I) • Low Binding Capacity • No Matrix Depth • Large/Moderate MW Molecules • Low Non-Specific Binding • High Binding Capacity • Flexible • Large/Moderate or Low MW Molecules • Minimal Non-Specific Binding = COOH Carboxyl Surfaces
- 11. Reichert Sensor Surfaces (II) • Steptavidin Amine Coupled • Capture Biotinylated Ligands • Stable Surface • Alkyl Surface • Capture Lipid Monolayer • Regenerate with CHAPS • Nitriloacetic Acetic Acid Surface • Capture HIS-Tagged Ligands • Decaying Surface • Regenerate with Imidazole • Protein A Amine Coupled • Capture IgGs • Regenerate with pH 2 buffer
- 12. Applications 1.) Traditional antibody-antigen and protein-small molecule kinetic and affinity data 2.) Cell adhesion and binding studies 3.) Cell-Protein interactions 4.) SPR-MS on-line coupling 5.) Liposome-Peptide binding
- 13. Example 1: Traditional Biomolecular Interaction Analysis (Antibody-Antigen and Protein-Small Molecule)
- 14. Antibody-Antigen 10000 15000 20000 25000 30000 35000 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Time (sec) Response(uRIU) EDC/NHS 1M Ethanolamine, pH 8.5 Anti-HSA in 10 mM NaOAc pH 5.2 Anti-HSA Immobilization 17900 18100 18300 18500 18700 18900 1000 2000 3000 4000 5000 6000 HSA Kinetic Run HSA Kinetic Global Fit
- 15. Inhibitor Kinetics 4-CBS Methane Sulfonamide KD = 857 nM KD = 650 µM
- 16. Example 2: Cell Adhesion Studies In collaboration with Dr. Michael Hill, Bioengineering Department, SUNY Buffalo
- 17. Cell Attachment Studies Flow rate reduced • Differential cell adhesion to immobilized proteins can be correlated to protein surface energy • Proteins with higher surface energy have greater tendency to bind cells Cells
- 18. Visualization of Cell Monolayers Collagen I Matrigel® Serum 200 µm 10x20x 200 µm
- 19. Hyper-Osmolar Shock Response • 100 mM hyperosmolar mannitol • Isotonic HEPES Collagen I Matrigel® Serum
- 20. Greater Strength of Binding on Collagen I 200 µm 200 µm Collagen I Matrigel® 10x20x Serum
- 21. • Collagen I: Higher γp and γ+ correlate with increased cell adhesion strength. This is the condition for ECs on stromal tissue as they undergo angiogensis. • Matrigel®: Lower γp and γ+ correlates with reduced EC interaction. This occurs when cells form monolayers on basement membranes • Young’s modulus of adhesion calculated via SPR • Matrigel® ~0.5MPa & Collagen ~2MPa • All results correlate well with AFM Conclusions (I) Collagen I: Immobilized arrays of Lewis acid and Lewis base groups Matrigel®: Cross-linked structure causes shielding of Lewis acid/base. + = Lewis acid - = Lewis base
- 22. • SPR: Used to probe cell-matrix interactions in the context of both specific and non-specific cell adhesion systems • The two types of adhesion often co-exist in particular biological contexts • SPR is useful to dissect intermolecular force characteristics of cell adhesion in different model systems, especially at short length and time scales • Hyperosmolar shock studies with SPR: can quantify strength of cell interactions with proteins • Cheaper, simpler, easier compared to past methods • Similar quantitative results in comparison to Atomic Force Microscopy Conclusions (II)
- 23. Example 3: Protein Binding to Living CHO Cells
- 24. Poly-l-lysine Immobilization Via Amine Coupling – 278 µRIU EDC/NHS 400 µg/mL Poly-l- Lysine in 10 mM Sodium Acetate pH 5.2 1 M Ethanolamine HCl pH 8.5
- 25. CHO K1 Cells Capture – 644 µRIU 50 µL stock/ mL CHO K1 Cells in HBS
- 26. Kinetic Titration Results with Fibrinogen ka = 5.114e5 M-1s-1 kd = 3.189e-2 s-1 KD = 62.4 nM 2.94 µM 1.47 µM 735 nM 368 nM 184 nM
- 27. • Results indicate that the measurement of cell adhesion can be easily performed using a Reichert SPR system. • In general, the study demonstrates the ability to perfuse live cells and probe cell-protein interactions in the time scale of typical SPR runs. • Reichert SPR systems provide the means to obtain quantitative measurements by enabling ready manipulation of cell capture flow rates, detachment kinetics under defined shear stress, and studies of osmotic pressure perturbation. • In comparison to other competing technologies like atomic force microscopy, SPR measurements are more straightforward and cost- effective and the measurements reflect the behavior of the average cell in a cell population (rather than individual cells). Conclusions from Cell Binding Studies
- 28. Example 4: SPR Coupling to Mass Spectrometry ?
- 29. SPR-Mass Spec Coupling Schematic
- 30. Horse Heart Myoglobin (HHM)/anti-HHM as Model System • SPR sensorgram obtained for HHM/anti-HHM in SPR/mass spec coupling interface • Total Ion chromatogram and multiple charged ions identify the unfolded apoprotein recognized by the antibody
- 31. Epitope Determination • Aß- autoantibody Immobilized on a Dextran sensor chip • Tryptic mixture of Aß-peptide fragments injected over immobilized antibody • Sensorgrams of the epitope peptide binding to Aß- autoantibody • ESI-MS identification by online SPR- MS of the epitope Aß(17-28) eluted from the Aß-antibody upon proteolytic extraction
- 32. Applications of SPR-MS Analyzer • Affinity-based biomarker evaluation • Identification of protein and peptide epitopes • Precise antibody affinity characterization • Direct label-free antigen quantification
- 33. Example 5: Annexin V and PSP1 Peptide Binding to PS Liposomes Annexin V – Popular probe for imaging apoptotic cell Peptide –based PS Indicator (PSP1) Kim S, Bae SM, Seo J, Cha K, Piao M, Kim S-J, et al. (2015) Advantages of the Phosphatidylserine-Recognizing Peptide PSP1 for Molecular Imaging of Tumor Apoptosis Compared with Annexin V. PLoS ONE 10(3): e0121171. doi:10.1371/journal.pone.0121171
- 34. Vesicle Capture 10000 10500 11000 11500 12000 12500 13000 13500 14000 14500 15000 10550 11550 12550 13550 14550 15550 Time (sec) Response(uRIU) Sample Channel Reference Channel 3260 uRIU of PS Vesicles on surface • Vesicles extruded through 100 nm membrane • Lipophilic surface preserves vesicle bilayer conformation
- 35. PSP1 and Annexin V Sensorgrams and Kinetic Data
- 36. The Reichert SPR Advantage • Your partner every step of the way – Unmatched customer service and support solutions – Maximum uptime drives better results • Solve your research bottlenecks – Scalable to research and lab needs – Systems accessible to your lab • Reliable binding, kinetics, concentration and thermodynamic data – Helping you answer questions quantitatively • Increase your sample flexibility – Broader application options – Robust fluidics • Reduce your equipment and maintenance costs