Liquid chromatography/tandem mass spectrometry characterization of oxidized amyloid beta peptides as potential biomarkers of Alzheimer’s disease Koichi Inoue 1 , Carlos Garner 2 , Bradley L. Ackermann 2 , Tomoyuki Oe 1 and Ian A. Blair 1 * 1 Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, 854 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104- 6160, USA 2 Drug Disposition, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA Received 6 December 2005; Revised 12 January 2006; Accepted 12 January 2006 Alzheimer’s disease is characterized by the deposition of senile plaques that consist primarily of amyloid b peptides. There is substantial evidence that amyloid b is oxidized in vivo, which has led to the suggestion that oxidative stress is an important mediator of Alzheimer’s disease. Metal-catalyzed oxidation can mimic in vivo oxidation of amyloid b because the metal ion binds to the amino acid residues at the site of oxidation, which then deliver reactive oxygen species to that site. Based on electrospray mass spectrometry, it has been suggested that metal-catalyzed oxidation occurs on histidines-13 and -14. Unfortunately, the amyloid b peptides provide complex spectra, so it is difficult to definitively characterize the sites of oxidation. Trypsin digestion of both native and oxidized amyloid b 1–16 and amyloid b 1–40 resulted in the formation of tryptic peptides corresponding to amyloid b 6–16 , which could be separated by liquid chromatography (LC). Sites of oxidation were then unequivocally characterized as histidine-13 and histidine-14 by LC/tandem mass spectrometric (MS/ MS) analysis of the tryptic peptides. The ability to analyze the specific amyloid b 6–16 tryptic fragments derived from full-length amyloid b peptides will make it possible to determine whether oxidation in vivo occurs at specific histidine residues and/or at other amino acid residues such as methionine-35. Using methodology based on LC/MS/MS it will also be possible to analyze the relative amounts of oxidized peptides and native peptide in cerebrospinal fluid from patients with Alzheimer’s disease as biomarkers of oxidative stress. Copyright # 2006 John Wiley & Sons, Ltd. Studies of subjects with early onset of Alzheimer’s disease (AD) have indicated that metabolism and/or modification of amyloid-beta (Ab) peptides is involved. 1 The Ab cascade hypothesis suggests that excessive plaque formation results from deposition of Ab peptides in the brain and that is an important step in the pathogenesis of AD. 2 Ab peptides are derived from the membrane-bound amyloid precursor protein (APP). 3–5 Normal catabolism of APP is known to involve cleavage by b-secretase and g-secretase, to produce Ab peptides with carboxyl-terminal heterogeneity. Evidence is also emerging which supports a role for metal ions and reactive oxygen species (ROS) in the pathogenesis of AD. 6–9 Longer forms of Ab, such as Ab 1–40 and Ab 1–42 (Scheme 1), are regarded as particularly pathogenic because they are overproduced as a result of familial gene mutations. 10,11 This has suggested that Ab peptides in cerebrospinal fluid (CSF) can be used as potential biomarkers of AD. 12,13 Many biomarker studies conducted with CSF have employed methodology based on enzyme-linked immuno- sorbent assay (ELISA) using antibodies against total Ab. 14,15 More recent studies have used specific ELISA assays for the quantitative analysis of each of the different Ab peptides in CSF. 16,17 Although some studies have found a slight decrease in CSF level of total Ab in AD, there is a large overlap between AD subjects and controls. Other studies have found no change in total CSF Ab for subjects with AD. 12–14,18–20 A number of studies showed that CSF Ab 1–42 levels, as determined by ELISA, were significantly decreased in AD subjects when compared with control groups. 17,18,21–24 In addition, CSF Ab 1–42 levels did not differ significantly between a normal control group and a mildly cognitively impaired group. 25 However, there was a marked reduction of CSF Ab 1–42 levels in disorders without Ab plaques, such as Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, and multiple system atrophy. 26–28 More specific methodology based on mass spectrometry (MS) has been used to a limited extent for the analysis of Ab peptides in CSF. 29–32 For example, Lewczuk et al. reported the existence of Ab 2–46 and Ab 1–45 in CSF using surface-enhanced laser desorption/ ionization time-of-flight (SELDI-TOF)-MS. 32 Yuan and Desiderio used MS-based methodology in a peptidomics approach to biomarker discovery in CSF. 33 Other CSF peptides have been analyzed as biomarkers of AD. For example, in one rigorous study, Clark et al. analyzed RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2006; 20: 911–918 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2395 *Correspondence to: I. A. Blair, Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, 854 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160, USA. E-mail: ian@spirit.gcrc.upenn.edu Contract/grant sponsor: National Institutes of Health; contract/ grant number: RO-1 CA91016. Copyright # 2006 John Wiley & Sons, Ltd.