Conformation-Specific Peptide Arrays Preparation of α-Helical Peptide Arrays on Self-Assembled Monolayer Surfaces using Soft-Landing and Reactive Landing of Mass-Selected Ions**

The α-helix – a common building block of the protein secondary structure – plays an important role in determining protein structure and function. The biological function of the α-helix is mainly attributed to its large macrodipole originating from the alignment of individual dipole moments of peptide bonds. Preparation of directionally aligned α-helical peptide layers on substrates has attracted significant attention because the resulting strong net dipole is useful for a variety of applications in photonics, 2 , 3 molecular electronics, and catalysis. 7 In addition, conformationally-selected α-helical peptide arrays can be used for detailed characterization of molecular recognition steps critical for protein folding, enzyme function and DNA binding by proteins. Existing technologies for the production of α-helical peptide surfaces are based on a variety of solution phase synthetic strategies 8 11 that usually require relatively large quantities of purified materials. Preparative mass spectrometry based on soft-landing (SL) 12 24 of mass-selected ions is a viable alternative to the existing surface modification approaches. It has been demonstrated that SL enables highly specific preparation of uniform thin films of biological molecules on substrates. 25 28 In addition, reactive landing (RL), in which SL is followed by covalent linking of molecules to chemically reactive surfaces, can be used for controlled immobilization of peptides and proteins on solid supports. 29 31 Because SL is a relatively gentle ion deposition technique it is easy to preserve the primary structure of deposited species. However, it is very difficult to control the secondary structure of soft-landed biomolecules because electrospray ionization (ESI) utilized in these experiments generates ions in a variety of different conformations. Here we present a first study focused on preparation of conformationally-selected peptide arrays using SL of mass selected peptide ions on self-assembled monolayer (SAM) surfaces. We selected the singly protonated Ac-A15K peptide as a model system for this study because ion mobility measurements and molecular dynamics (MD) simulations demonstrated that this peptide forms a very stable α-helical conformation in the gas phase stabilized by the interaction between the protonated C-terminal lysine residue and the dipole of the helix. 35 We demonstrate formation of the α-helical peptide array on an inert SAM of alkylthiol on gold (HSAM) and covalent immobilization of the Ac-A15K peptide on a reactive SAM of N-hydroxysuccinimidyl ester terminated alkylthiol on gold (NHS-SAM) with retention of the secondary structure. Because the NHS-SAM surface readily reacts with primary amino groups in proteins or peptides by forming amide bonds this substrate has been previously used for efficient covalent immobilization of softlanded peptides onto SAMs via the formation of an amide bond between the SAM and the amino group of the lysine side chain. Experiments were performed using an ion deposition apparatus described in detail elsewhere. The instrument is equipped with a high-transmission electrospray ionization (ESI) source and a quadrupole mass filter. Singly protonated peptide molecules, [AcA15K+H] (m/z = 1254.6), produced by ESI were mass-selected and deposited onto SAM surfaces at the collision energy of 20 eV. Inert SAMs of dodecanethiol (HSAM) and reactive NHS-SAMs on gold were used as SL targets. The deposition time was controlled by monitoring the ion current on the surface. Typical SL experiments reported in this study correspond to deposition of ca. one monolayer of the peptide onto the surface. ESI deposition was performed by placing the target in front of the ESI emitter tip for controlled time duration. Secondary structure of the peptides in the electrospray solution (76μM Ac-A15K in 50:50 (v/v) methanol/water with 1% acetic acid) was determined using circular dichroism (CD) spectroscopy. Infrared reflection absorption spectroscopy (IRRAS) and time of flight-secondary ion mass spectrometry (TOF-SIMS) were used to obtain structural information for soft-landed species. IRRAS is used for characterization of the secondary structure of peptides on SAM surfaces based on the presence and position of amide I, amide II and amide A bands originating from peptide bonds. The amide I band, commonly used for characterization of the peptide secondary structure using FTIR, is dominated by the C=O stretching vibrations of amide groups and gives rise to infrared absorption in the region between 1600 and 1700 cm. The amide II band represents mainly N-H bending (60%) with some C-N stretching (40%) and usually appears at ~ 1550 cm. Although fairly abundant, the amide II band is not very sensitive to changes in the peptide secondary structure. The amide A band responsible for absorption in the 3200-3300 cm region corresponds to the stretching mode of the N-H bond. Because the N-H stretching vibration is strongly affected by the hydrogen bonding environment, this band is also sensitive to changes in the secondary structure. [∗] Dr. P. Wang, Dr. J. Laskin Fundamental Sciences Division Pacific Northwest National Laboratory P.O.Box 999 K8-88 Richland, WA 99352 USA Fax: (+1) 509-371-6139 E-mail: [email protected] Homepage: Hhttp://emslbios.pnl.gov/id/laskin_j Figure 1 compares standard CD spectra of the α-helix, β-sheet and random coil with the spectrum obtained for the ESI solution of Ac-A15K. The CD spectrum of the Ac-A15K solution shows the presence of a mixture of conformations dominated by the β-sheet and a small fraction of the α-helix and random coil. Similarly, the IRRAS spectrum of the Ac-A15K layer prepared on the HSAM surface using ESI deposition is also dominated by the characteristic features of the β-sheet structure. Specifically, the IRRAS spectrum [∗∗] This work was supported by the Laboratory Directed Research and Development Program at Pacific Northwest National Laboratory and the grant from the Chemical Sciences Division, Office of Basic Energy Sciences of the U.S. Department of Energy. The authors thank Dr. John Cort for technical assistance and helpful discussions.