Analyzing Protein Sorting at Surfaces

David W. Grainger, Ph.D.

Eccles Presidential Endowed Chair and Professor

Departments of Pharmaceutics and Pharmaceutical Chemistry, and Bioengineering

University of Utah, Salt Lake City, UT 84112-5820 USA (david.grainger@utah.edu)

Immobilized proteins are of increasing technological interest in numerous applications.  Methods to analyze them in situ on surfaces are problematic. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is very sensitive for surface analysis (10nm sampling depth) and is useful for trace element detection. With the advent of improved analyzers, imaging ToF-SIMS provides spatial distribution of different species to yield surface reactivity maps. Use of multivariate analysis, especially Principal Component Analysis (PCA), makes this technique even more powerful by differentiating regions with different chemistries. ToF-SIMS and PCA have been used to study a commercial, patterned polymer chemistry with applications in diagnostics, assays, bio-chips and cell-based biosensors.(1) The chemistry is based on N-hydroxysuccinimide (NHS) esters, widely used as leaving groups to activate covalent coupling of amine-containing biomolecules onto surfaces. Our previously studied model system -- NHS molecules self-assembled on gold -- and XPS and ToF-SIMS were used to characterize and understand the NHS hydrolysis and regeneration at surfaces (2) on commercial poly(ethylene glycol) (PEG)-based reactive films on glass slides. Photolithographically patterned NHS were imaged with ToF-SIMS/PCA. NHS surface reactive zones are clearly resolved at high sensitivity despite the complexity of the polymer matrix chemistry. Surface-specific protein coupling was observed by surface-selective reaction of streptavidin with the NHS patterns.(3) Patterns of two coplanar affinity ligands (biotin and chloroalkane) were used for specific immobilization of two different proteins (streptavidin and HaloTag® enzyme). Spontaneous formation of high-fidelity surface patterns of the two proteins from their mixed solution was observed and characterized, a process termed “surface self-selection’.(4) ToF-SIMS amino acid-derived ion fragment yields summed to produce surface images can reliably correlate patterned surface regions to bound proteins, but cannot readily discriminate different proteins.  However, PCA of ToF-SIMS data improves discrimination of ions specific to each protein, facilitating surface pattern discrimination based on protein type.  ToF-SIMS imaging also detects regions where trace UV-exposed photoresist residue remained.(5,6)   Finally, we have used TOF-SIMS PCA to discriminate antibody orientation states immobilized at surfaces.(7)

References:

1. G. Harbers et al. Chem. Mater. 19, 4405-4414 (2007); 2. Cheng, F., et al., Anal. Chem., 2007. 79 8781-8788; 3. Takahashi, et al. Adv. Func. Mat. 2008. 18, 2079-2088; 4. M. Dubey, et al. Adv. Funct. Mater., 19; 1–10 (2009); 5. M. Dubey, et al., Surf. Interfacial Anal., 41: 645–652 (2009); 6. M. Dubey, et al., Surf. Interface Anal., 2011; 7. F. Liu et al., Anal. Chem., 82, 2947–2958 (2010).