title: Multi-technique Characterization of Adsorbed Peptide and Protein Orientation: LK310and Protein G B1.
abstract: The ability to orient biologically active proteins on surfaces is a major challenge in the design, construction, and successful deployment of many medical technologies. As methods to orient biomolecules are developed, it is also essential to develop techniques that can an accurately determine the orientation and structure of these materials. In this study, two model protein and peptide systems are presented to highlight the strengths of three surface analysis techniques for characterizing protein films: time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum-frequency generation (SFG) vibrational spectroscopy, and near-edge x-ray absorption fine structure (NEXAFS) spectroscopy. First, the orientation of Protein G B1, a rigid 6 kDa domain covalently attached to a maleimide-functionalized self-assembled monolayer, was examined using ToF-SIMS. Although the thickness of the Protein G layer was similar to the ToF-SIMS sampling depth, orientation of Protein G was successfully determined by analyzing the C2H5S+intensity, a secondary ion derived from a methionine residue located at one end of the protein. Next, the secondary structure of a 13-mer leucine-lysine peptide (LK310) adsorbed onto hydrophilic quartz and hydrophobic fluorocarbon surfaces was examined. SFG spectra indicated that the peptide's lysine side chains were ordered on the quartz surface, while the peptide's leucine side chains were ordered on the fluorocarbon surface. NEXAFS results provided complementary information about the structure of the LK310film and the orientations of amide bonds within the LK310peptide.
authors: National ESCA and Surface Analysis Center for Biomedical Problems, Department of Chemical, University of Washington, Seattle, WA 98195.
authors: J E
authors: JE
authors: Baio
authors: T
authors: T
authors: Weidner
authors: N T
authors: NT
authors: Samuel
authors: Keith
authors: K
authors: McCrea
authors: Loren
authors: L
authors: Baugh
authors: Patrick S
authors: PS
authors: Stayton
authors: David G
authors: DG
authors: Castner
keywords: LK310 peptides; NEXAFS; Protein G B1; SFG; ToF-SIMS; biointerfaces; hexafluoropropylene; maleimide-oligo(ethylene glycol); peptides; proteins
references: Anal Chem. 2007 Jun 15;79(12):4390-400
references: 17492838
references: Anal Chem. 2008 Apr 1;80(7):2564-73
references: 18302347
references: Biophys J. 1990 May;57(5):965-75
references: 2160300
references: Phys Rev Lett. 1994 Jan 10;72(2):238-241
references: 10056094
references: Anal Chem. 1993 May 15;65(10):1431-8
references: 8517550
references: J Am Chem Soc. 2008 Oct 1;130(39):13033-9
references: 18774819
references: Bioconjug Chem. 2006 Nov-Dec;17(6):1592-600
references: 17105240
references: Chem Commun (Camb). 2008 Oct 7;(37):4400-12
references: 18802573
references: Appl Spectrosc. 2008 Sep;62(9):937-40
references: 18801230
references: Mass Spectrom Rev. 2008 Jul-Aug;27(4):289-315
references: 18421766
references: Biointerphases. 2010 Mar;5(1):9-16
references: 20408730
references: Chem Rev. 2006 Apr;106(4):1140-54
references: 16608175
references: Proc Natl Acad Sci U S A. 2005 Apr 5;102(14):4978-83
references: 15793004
references: Anal Chem. 2007 Nov 15;79(22):8781-8
references: 17929879
references: Anal Bioanal Chem. 2008 Jul;391(5):1521-44
references: 18504563
references: Anal Chem. 2003 Sep 15;75(18):4740-6
references: 14674449
references: Science. 1989 Feb 3;243(4891):622-8
references: 2464850
references: Langmuir. 2005 Mar 29;21(7):2662-4
references: 15779931
references: J Biomed Mater Res A. 2003 Jan 1;64(1):1-11
references: 12868435
references: J Biomed Mater Res A. 2003 Oct 1;67(1):179-90
references: 14517875
references: Langmuir. 2004 Mar 2;20(5):1877-87
references: 15801458
references: J Am Chem Soc. 2006 Mar 22;128(11):3598-607
references: 16536533
references: Biointerphases. 2006 Jun;1(2):82-92
references: 20408620
references: J Am Chem Soc. 2003 Mar 12;125(10):3150-8
references: 12617683
references: J Electron Spectros Relat Phenomena. 2009 May 1;172(1-3):2-8
references: 20161353
references: J Biomed Mater Res. 2001 Dec 5;57(3):432-40
references: 11523038
references: J Phys Chem B. 2009 Nov 26;113(47):15423-6
references: 19873996
references: Angew Chem Int Ed Engl. 2004 Feb 27;43(10):1246-9
references: 14991789
references: J Am Chem Soc. 2006 Jan 11;128(1):2-3
references: 16390092
references: Biomaterials. 2003 May;24(11):1897-908
references: 12615480
references: Anal Chem. 2007 Feb 15;79(4):1377-85
references: 17297937
references: J Biomed Mater Res. 1998 Jul;41(1):95-103
references: 9641629
references: Chem Rev. 2002 Aug;102(8):2693-724
references: 12175265
references: J Phys Chem B. 2009 Aug 20;113(33):11550-6
references: 19639981
references: Langmuir. 2005 Dec 20;21(26):12327-32
references: 16343010
references: Anal Chem. 1997 Nov 15;69(22):4601-7
references: 9375518
references: Phys Rev Lett. 2008 Mar 7;100(9):096102
references: 18352727
references: Anal Bioanal Chem. 2009 Mar;393(5):1407-16
references: 18807017
references: Annu Rev Phys Chem. 1989;40:1-29
references: 18338977
references: Langmuir. 2010 Mar 2;26(5):3433-40
references: 20175575
references: Biophys J. 1995 Jun;68(6):2573-9
references: 7647260
references: Biomaterials. 2003 Sep;24(21):3635-53
references: 12818535
references: J Phys Chem B. 2007 Jul 5;111(26):7691-9
references: 17559260
references: Langmuir. 2010 Nov 2;26(21):16434-41
references: 20384305
references: J Biomater Sci Polym Ed. 2008;19(6):725-53
references: 18534094
references: J Am Chem Soc. 2002 Jun 5;124(22):6297-303
references: 12033857
references: J Am Chem Soc. 2009 Oct 14;131(40):14148-9
references: 19764755