Notes & Tips High-throughput screening in two dimensions: Binding intensity and off-rate on a peptide microarray Matthew P. Greving a,b , Paul E. Belcher a , Conor D. Cox a , Douglas Daniel a , Chris W. Diehnelt a , Neal W. Woodbury a,b, * a The Biodesign Institute at Arizona State University, 1001 S. McAllister Avenue, Tempe, AZ 85287-5201, USA b Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA article info Article history: Received 4 December 2009 Received in revised form 2 March 2010 Accepted 2 March 2010 Available online 6 March 2010 abstract We report a high-throughput two-dimensional microarray-based screen, incorporating both target bind- ing intensity and off-rate, which can be used to analyze thousands of compounds in a single binding assay. Relative binding intensities and time-resolved dissociation are measured for labeled tumor necro- sis factor alpha (TNF-a) bound to a peptide microarray. The time-resolved dissociation is fitted to a one- component exponential decay model, from which relative dissociation rates are determined for all pep- tides with binding intensities above background. We show that most peptides with the slowest off-rates on the microarray also have the slowest off-rates when measured by surface plasmon resonance (SPR). Ó 2010 Elsevier Inc. All rights reserved. Binding affinity, typically reported as the dissociation constant (K d ), consists of the ratio of the dissociation rate (off-rate, k off ) and the association rate (on-rate, k on ). Dissociation constants are commonly determined using steady-state binding measurements, leaving the kinetic details unknown. Often steady-state affinity alone is not sufficient, and the binding kinetics must be deter- mined to fully understand the binding mechanism or to compre- hensively screen a library of potential ligands for the desired activity. With the advent of surface plasmon resonance (SPR) 1 [1–3], determining binding kinetics has become much more straight- forward. Currently SPR can be used to simultaneously monitor bind- ing kinetics of a few hundred arrayed compounds [3–6], which is small when compared to high-density microarrays [7–12]. There- fore, analyzing many thousands of interactions can be time consum- ing if not impossible to do with SPR. In addition to SPR, other technologies exist for determining binding kinetics on an array [13–16]. However, these require custom array substrates or instru- mentation. A method that rapidly screens both binding levels and kinetics of thousands of interactions using commercially available substrates and instrumentation would be very useful because, from this screen, a much smaller number of compounds could be selected for quantitative kinetic analysis using SPR. Here, we describe a fluo- rescence-based method for screening a library of compounds on a microarray. Both target binding levels and off-rates are measured in a single experiment using commercially available instruments [17,18], substrates [19–21], and fluorophores [22]. Recognizing both the analytical strengths and the throughput limitations of SPR, the method described here utilizes microarrays to rapidly screen a peptide library for sequences with both signif- icant binding and slow off-rates against the target protein tumor necrosis factor alpha (TNF-a). From this screen, a more manage- able number of peptides are selected for quantitative kinetic mea- surements with SPR. To test this approach, a peptide library containing more than 800 unique sequences was printed as a high-density microarray of approximately 2500 features that are each 100 lm in diameter, including replicates and internal controls. In addition to binding levels, TNF-a dissociation rates were observed on the peptide microarray in real time. TNF-a dissociation rates were chosen be- cause of the importance of off-rates in binding a target in the pres- ence of a large amount of competitor, particularly when used in molecular diagnostic applications [23,24]. However, this method is not limited to off-rates and could also be applied to screening on-rates or both on-rates and off-rates on a microarray. To increase the availability and ease of implementation of this approach, peptides were printed with a commercial contact printer on aminopropyltriethoxysilane (APTES)-coated 7.5  2.5 cm micro- scope slides using standard attachment chemistry. APTES surfaces were treated with sulfo-SMCC, which contains an N-hydroxy- succinimide (NHS) group that reacts with the amine on the glass surface to form a covalent attachment and an unreacted maleimide group. All peptides printed on the microarray contained a C-terminal cysteine, which reacts with the maleimide group on the surface, thereby covalently immobilizing all peptides with the same 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.03.002 * Corresponding author at: The Biodesign Institute at Arizona State University, 1001 S. McAllister Avenue, Tempe, AZ 85287-5201, USA. Fax: +1 480 727 8305. E-mail address: nwoodbury@asu.edu (N.W. Woodbury). 1 Abbreviations used: APTES, aminopropyltriethoxysilane; SPR, surface plasmon resonance; TNF-a, tumor necrosis factor alpha. Analytical Biochemistry 402 (2010) 93–95 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio