DOI: 10.1021/la101253g 12075 Langmuir 2010, 26(14), 12075–12080 Published on Web 06/08/2010 pubs.acs.org/Langmuir © 2010 American Chemical Society Detecting the Presence of Denatured Human Serum Albumin in an Adsorbed Protein Monolayer Using TOF-SIMS Ivan M. Kempson,* ,†,‡ Amanda L. Martin, ‡ John A. Denman, ‡ Peter W. French, § Clive A. Prestidge, ‡ and Timothy J. Barnes ‡ † Institute of Physics, Academia Sinica, Nankang, Taipei 115, Taiwan, ‡ Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA, 5095, Australia, and § Fermiscan Ltd, 48 Hunter St, Sydney, NSW, 2000, Australia Received March 30, 2010. Revised Manuscript Received May 29, 2010 We demonstrate the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in conjunction with multivariate statistics to differentiate trace levels of denatured proteins in adsorbed monolayers; specifically, human serum albumin (HSA) on oxidized silicon substrates. Subtle differences in protein conformation due to thermal denaturation of HSA, unable to be determined by dynamic light scattering nor circular dichroism, were differentiated by TOF-SIMS. The fragmentation pattern is highly sensitive to protein conformation, allowing assessment of relative amounts of proteins in mixtures and quantifying amounts of denatured protein in a sample. Discussion is presented on ascribing orientation and conformational differences between samples based upon TOF-SIMS spectra. This has implications for detecting denatured protein in biotechnology and medical applications. 1. Introduction Protein structure and functionality are highly sensitive to local environment. Subtle changes can occur due to effects of disease and bodily dysfunction. Additionally, such sensitivity has severe implications on quality control in manufacturing and purification processes inducing undesirable effects. Minor structural changes can alter protein functionality which is critical to avoid, for instance, in drug delivery systems. 1,2 Circular dichroism (CD) provides a common experimental approach to study protein secondary structure and interactions. 3-6 While CD is highly informative, alternative techniques may offer greater sensitivity to monitor protein integrity, orientation and interactions. Changes in protein structure and conformation can be induced by various health disorders, e.g. analysis of blood serum has identified solution-phase structural changes due to cancer. 7 The conformational changes lead to increasing interactions between water molecules and polar functional groups, with greater dis- turbance occurring as the disease progresses. In the solid phase, changes in lyophilized serum samples were detected from cirrhosis and hepatocellular carcinoma patients. 8 The progression of the carcinoma results in phenotypic changes in proteins and other structural alterations in proteins produced in the liver. These differences were attributed to protein tertiary structural changes. Differences in blood serum due to ovarian cancer, prostate cancer, prostate inflammation, mammary cancer and rheumatic inflammation from healthy subgroups have also been detected. 9 The ability of TOF-SIMS to study any spectrometric frag- ment provides powerful opportunities to analyze organic sub- stances, for example, in cellular systems, 10 pharmaceuticals, 11-13 biomonitoring 14 and biomedical devices. 15 This technique coupled with multivariate principal component analysis 16-18 enables intricate analysis of subtle changes in organic materials. Several very interesting studies have been conducted with TOF- SIMS assessing protein adsorption with respect to surface com- position and subsequent induced conformational changes. 19-21 However, applications to probe protein orientation are in their infancy and major studies reporting quantitative differentiation of priorly and subtly denatured proteins in biologically relevant protein mixtures are not apparent. With these observations in mind, TOF-SIMS was explored for its ability to semiquantitatively differentiate protein mixtures based on concentration ratios and relative amounts of denatured components. TOF-SIMS coupled with multivariate statistical *Author to whom correspondence should be addressed at the Institute of Physics, Academia Sinica, Nankang, Taipei 115, Taiwan. Telephone: þ886 (0)3 578 0281. Fax: þ886 (0)3 578 3805. E-mail: Ivan.m.k@hotmail.com. (1) Prestidge, C. A.; Barnes, T. J.; Lau, C.-H.; Barnett, C.; Loni, A.; Canham, L. Expert Opin. 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Pharmacol. 2007, 59, 251. (12) Prestidge, C. A.; Barnes, T. J.; Mierczynska-Vasilev, A.; Kempson, I. M.; Peddie, F.; Barnett, C. Phys. Status Solidi A 2008, 205, 311. (13) Kempson, I. M.; Barnes, T. J.; Prestidge, C. A. J. Am. Soc. Mass. Spectr. 2010, 21, 254. (14) Kempson, I. M.; Skinner, W. M. Sci. Total Environ. 2005, 338, 213. (15) Aoyagi, S.; Takesawa, A.; Yamashita, A. C.; Kudo, M. Appl. Surf. Sci. 2006, 252, 6697. (16) Deslandes, A.; Jasieniak, M.; Ionescu, M.; Shapter, J. G.; Fairman, C.; Gooding, J. J.; Hibbert, D. B.; Quinton, J. S. Surf. Interface Anal. 2009, 41, 216. (17) Jasieniak, M.; Suzuki, S.; Monteiro, M.; Wentrup-Byrne, E.; Griesser, H. J.; Grøndahl, L. Langmuir 2009, 25, 1011. (18) Denman, J. A.; Kempson, I. M.; Skinner, W. M.; Kirkbride, K. P. Forensic Sci. Int. 2008, 175, 123. (19) Henry, M.; Bertrand, P. Surf. Interface Anal. 2009, 41(2), 105. (20) Henry, M.; Dupont-Gillain, C.; Bertrand, P. Langmuir 2003, 19, 6271. 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