Protein sorting at surfaces: Parallel affinity capture to patterns

Transcription

1 Protein sorting at surfaces: Parallel affinity capture to patterns Fang Cheng, Manish Dubey, Lara Gamble, David Castner University of Washington, USA Hironobu Takahashi, Fang Liu, Greg Harbers, David Grainger University of Utah, USA Biointerface Atlanta, October, 2010 NIBIB

2 Bulk adsorption reaction: Bio-immobilization Strategies Buffer Spot printing: quasiequilibrium hours, days Two distinct endpoints: - different densities - different efficiencies - different bioactivities - different physical states - different surface interactions nanoliter drop 2-3 seconds H 2 O Nonequilibrium dried DNA or protein spot 150 µm Glass Slide Glass Slide

3 EX 1: DNA Macrospots vs. Microarray spots: size matters! Higher DNA Density on Microarray spots because of rapid drying P 2p atomic % Microarray Macrospot (hand printed) Spotted bulk DNA concentration (µm) 100µm 5 mm P. Gong, G.M. Harbers, D.W. Grainger, Analy. Chem (2006) Immobilized DNA density % DNA target capture Maximum capture

4 Example 2: Micro-printed antibody arrays: printed dried proteins don t need covalent chemistry! Cy5: streptavidin print fluorescence 2500 Cy3: after antibody capture assay RFUs RFUs b SA SA-SH b SA SA-SH pre-blocked control activated slide SA: streptavidin 0 b SA SA-SH b SA SA-SH pre-blocked control activated slide SA-SH: thiolated streptavidin Blocked, inactivated surfaces No substantial difference between spotted proteins: non-thiolated streptavidin and thiolated streptavidin blocked and unblocked maleimide-modified surfaces Thiolreactive maleimide surfaces Wu et al., Biosen. Bioelectron., 2006

5 Commercial Functionalized PEG-based Bioassay Surface Chemistry N-hydroxysuccinimide (NHS): Reactive with amines in matrix Poly(ethylene glycol) (PEG): base film polymer G. M. Harbers et al., Chem. Mater., 19, 4405 (2007) Functionalized Non-Fouling Matrix Slide H (Schott-Nexterion): only PEG-based commercial coating

6 NHS Patterning on non-fouling PEG-based Surfaces using Photolithography H. Takahashi et al, Adv. Funct. Mater., , 2008 Using photolithography, NHS groups partially methoxylated NHS groups patterned on non-fouling PEG film background Photomask

7 Time-of-Flight Secondary Ion Mass Spectrometry: Chemical Mapping Mass spectrometer Image generation Time-of-flight tube Ion source Non-destructive (static) Primary ions (dose ~10 11 ions/cm 2 ) secondary ions from surface Positive Ion Spectrum Mass spec of surface zone ~ 1nm depth spatial mapping of chemistry low detection limits complex spectra (~10 4 peaks) Intensity PCA processing Energy (ev)

8 Protein Patterning: ToF-SIMS vs. Fluorescence Imaging ToF-SIMS PC1 Scores & Loadings: Patterned Protein Imaging Streptavidin/biotinylated BSA with Alexa555 Protein conjugation through NHS reaction Protein Patterning Amino acids! Amino acids Correlated patterns: ToF-SIMS image matches microscopy Fluorescence from NHS region but not from MeO region

9 Cell Pattern Fidelity in Serum on RGD peptide-patterned surface before RGD modification Ion images: RGD patterning RGD Cell culture in serum after RGD modification 3T3 cells adhere and grow only on NHS patterned + peptide patterned regions >48h incubation in serum, cells remain within the patterns On narrowest line pattern: cells spread and orient along pattern axis and proliferate within patterns Images taken at 24 h (a) and 48 h (b-e) after 3T3 seeding in serum Scale bar: (a-d) 200 µm, (e) 50 µm H. Takahashi et al, Adv. Funct. Mater., 2008

10 Protein self-sorting? Protein mixtures Expose to patterned surface Rinsed

11 Protein self-sorting! Dubey et al., Adv Funct. Mater, , Streptavidin 66kDa HaloTag 34kDa

12 Mixed HaloTag enzyme and streptavidin Dubey et al., Adv Funct. Mater, , Photo-copatterned biotin/chloroalkane Affinity captured HaloTag enzyme vs. streptavidin: side-by-side fluorescent microscopy imaging

13 ToF-SIMs Image contrast: composition differences in patterned proteins streptavidin HaloTag PC1 loadings plot for ToF-SIMS spectra regenerated for amino acid-specific ion fragments after protein sorting/immobilization. Cys + Met load in the HaloTag regions Thr, Trp, Tyr load in the streptavidin regions predicted from protein compositions shown here Possibility to chemically map these distinct proteins on surfaces??

14 Amino acid surface imaging Dubey et al., Adv Funct. Mater, , HaloTag Streptavidin Protein-surface selectivity: normalized ToF- SIMS images of protein-specific ion fragments PCA loadings plots: (a) Cys + Met ions (HaloTag) (b) Tyr + Thr + Trp ion fragments (streptavidin) BC patterned dual ligand surface exposed to a mixture of streptavidin and HaloTag proteins ToF-SIMS surface images summing all amino acid ion fragments for ligand-immobilized patterns: BM: (biotin/methoxy) MC (methoxy/chloroalkane) BC (biotin/chloroalkane) patterned hydrogel surfaces exposed to: streptavidin (SA), HaloTag (HT), and Mixed streptavidin and HaloTag (MIX)

15 Bio-immobilization imaging and tracking tools: Who really cares? Consider antibody arrays for diagnostics: Antibody array markets: $billions markets for clinical and biodefense analytes Less than 10% of immobilized antibodies on microarrays are bioactive: surface issue. Detection limits are generally pg/ml in buffer, many analytes are below this in serum. Improve assay performance by controlling antibody orientation/density??

16 Antibody orientational analysis by ToF-SIMS Hypothesis: sampling depth of ToF-SIMS is ~1-2 nm -- differentiates between 2-state (up-down) antibody-surface orientations 16 nm Protein A-antibody binding by F c interaction F ab 12 nm Design 1: Antibody binds protein A through F c fragment so F ab domain should be exposed in the protein A-patterned regions, detected by ToF-SIMs Protein A capture F c Design 2: Antibody binds surface hapten, fluorescein, via F ab fragments so F c domain should be exposed in fluorescein patterns by ToF-SIMS anti-fluorescein capture

17 Promoting and proving antibody orientation: protein A/anti-fluorescein : protein A : fluorescein hapten antibody immobilizes through protein A - antibody F c interaction Since antibody is an anti-fluorescein antibody, F ab region to interact with bound fluorescein

18 Amino acid composition for F(ab ) 2 and Fc fragments for murine antibody 2 Amino acid F(ab ) 2 [4FAB] Fc [1IGT] # residues % comp # residues % comp Ratio of Fab /Fc Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Data deposition: (PDB ID code 4FAB, 1IGT)

19 Is there an orientation dependence? Case 1: F ab /F c = 1.8 for Gly ToF-SIMS high Gly signal low Gly signal sampling depth Protein A-bound surface Fluorescein hapten-bound ToF-SIMS PCA peak loads positively in the Protein A F ab region

20 Is there an orientation dependence? Case 2: F ab /F c = 0.48 for His low His signal high His signal sampling depth Protein A-bound surface Fluorescein hapten-bound Due to orientation, the His fragment should load high for the Fc oriented patterns

21 Is there an orientation dependence? Case 3: F ab /F c = 1.03 for Ala same Ala signal same Ala signal sampling depth Protein A bound surface Fluorescein hapten Despite orientation, the Ala fragment loads the same for both regions: no image contrast for Ala imaging

22 Antibody orientation dependence ToF-SIMs sampling zone: 1-2nm deep Fc F(ab)2 Amino Acid tails-up vs heads-up F(ab)2 substrate Fc F ab /F c PCA loading region Cys 0.70 Fluorescein His 0.42 Fluorescein Pro 0.53 Fluorescein Leu 1.57 Protein-A Arg 1.21 Protein-A Gln 1.18 Protein-A

23 Fluorescence images: patterned protein-immobilized hydrogel surfaces protein A Anti-fluorescein antibody on protein A Anti-fluorescein Fab on fluorescein fluorescein Control Fab on protein A Control Fab on fluorescein Fc on protein A Fc on fluorescein Liu et al., Anal. Chem., 2010, 82, DOI /ac902964q.

24 Immobilized F ab and F c fragment controls: ToF-SIMS PCA Murine antibody pepsin-digested into F(ab) 2 and Fc fragments by pepsin Each fragment purified and immobilized on separate hydrogel surfaces ToF-SIMS spectra (n=3) were collected and processed with principal component analysis (0.36) (0.48) (0.71) F c controls (1.81) (1.6) (1.03) (1.9) F ab controls (1.3) ToF-SIMS PCA analysis consistent with the composition of F ab and F c

25 Affinity-immobilized antibody surface images and pattern contrasts generated from ToF-SIMS surface analysis data (a) Chemical image: all amino acids specific for Fab versus Fc domains (b) Chemical image; sum of all ToF-SIMS amino acid fragments Antibody orientational chemical surface mapping Liu et al., Anal. Chem., 2010, 82, DOI /ac902964q.

26 Patterned surfaces and chemical mapping: Antibody orientation Protein A/Fluorescein patterns react specifically to orient antibodies ToF-SIMS total ion images generated and PCA used to separate fragments for antibodies into different loadings for each orientation PCA loadings were mapped onto the surface for various amino acids Fluorescein patterns Asn Protein A patterns Gly +Trp +Tyr

27 Summary Surface-specific protein coupling by surface-selective simultaneous reactions of streptavidin with biotin patterns, and HaloTag specific for chloroalkane patterns. Protein self-sorting: high signal, low noise Antibody affinity immobilization: heads-up and heads-down orientation by sorting Chemical imaging can map antibody orientation on polymer surfaces

28 Acknowledgments Castner, Gamble and Grainger research groups Dr. S. Golledge, CAMCOR, Univ. Oregon Accelr8 Technology, Denver, CO Funding: NIH grants EB and EB , National Institute of Biomedical Imaging and Bioengineering, USA NIBIB Na + Image Produced by a Bi DC Beam Field of View: 150µm x 150µm

29 Principal component analysis (PCA) for ToF-SIMS A multivariate analysis method, reducing a large set of correlated variables to a smaller number of uncorrelated variables called principal components (PCs). Rapid decomposition of ToF- SIMS data into two dimensionless matrices: scores and loadings. Correlation of spectral information to interpret images PCA schematic: D. Graham

30 Protein sorting: dual ligands, selective affinity binding

31 Fab and Fc fragments immobilized on polymer and gold-nhs surfaces PC-1 and PC-2 scores plot from ToF-SIMS amino acid data for Fab and Fc fragments PC-1 distinguishes substrate surface compositional differences (i.e., gold vs. polymer) PC-2 distinguishes Fc fragments from Fab fragment chemistry PCA loadings plot for PC-2 for amino acid fragments derived from ToF-SIMS analysis of Fab and Fc fragments Fc fragments load in positive direction Fab fragments load in the negative direction Results correlate well with amino acid compositions in each domain

32 ToF-SIMS surface images summing all amino acid ion fragments for ligandimmobilized high-fidelity patterns exposed to antibody protein A/methoxy-capped methoxy-capped/fluorescein protein A/fluorescein Image contrast from ion yield contrasts across the patterns: orientation

33 Protein self-sorting: streptavidin and HaloTag selecting surface-immobilized biotin and chloroalkane ligands Dubey et al., Adv Funct. Mater, , 3046.