Oxidation of methionine 35 attenuates formation of amyloid beta -peptide 1-40 oligomers.

Amyloid plaques formed by aggregation of the amyloid beta-peptide (Abeta) are an intrinsic component of Alzheimer disease pathogenesis. It has been suggested that oxidation of methionine 35 in Abeta has implications for Alzheimer disease, and it has been shown that oxidation of Met-35 significantly inhibits aggregation in vitro. In this study, the aggregational properties of Abeta-(1-40) before and after Met-35 oxidation were investigated using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The results show that Abeta-(1-40)Met-35(O) trimer and tetramer formation is significantly attenuated as compared with Abeta-(1-40). This suggests that oxidation of Met-35 inhibits a conformational switch in Abeta-(1-40) necessary for trimer but not dimer formation. Random incorporation of Abeta-(1-40) and Abeta-(1-40)Met-35(O) in homo- and heterooligomers could also be observed. This is the first report of an early rate-limiting step in Abeta-(1-40) aggregation. Slowing of the fibrillization process at this early step is likely to support prolonged solubility and clearance of Abeta from brain and may reduce disease progression.

Amyloid ␤-peptide (A␤) 1 is a 39 -43 amino acid long peptide derived from a larger transmembrane protein, the amyloid precursor protein. A␤ is the central component of neuritic plaques in Alzheimer disease (AD) brain. A large number of studies have focused on the structure, aggregational properties and fibril formation (1)(2)(3)(4)(5), and neurotoxicity (6,7) of A␤ peptides and their roles in AD.
Chemical analysis of human neuritic plaques reveals a complex mixture of chemically altered A␤ deposited along with glycoproteins, glycolipids, and other ancillary components (3). Oxidized methionine, in position 35 (Met-35) of A␤, methionine sulfoxide (Met-35(O)), has been detected in samples extracted from post-mortem AD plaques (8). These findings have yet to be extended, eradicating the possibility of a post-mortem delay or a preparatively induced oxidation artifact before in vivo extrapolation can be made. Few studies have explored the consequences of Met-35 oxidation on peptide chemical characteristics or biological actions. There are, however, some intriguing findings and some contradicting reports. Substituting synthetic A␤1-42 with methionine sulfoxide Met-35(O) results in enhanced A␤1-42-mediated cellular toxicity (9,10). Whether these actions are a consequence of attenuated (11) or accelerated (10) fibril formation remains to be determined. A␤-(1-40)Met-35(O) is dramatically less prone to aggregation and fibril formation as compared with A␤- , shown in circular dichroism studies (12). This is also reflected by the inhibition of conformational switching of A␤-(1-40)Met-35(O) from random coil to ␤-sheet, observed using millimolar peptide concentrations and NMR (12). The inhibited aggregation can, however, be overcome by increasing the concentration of A␤-(1-40)Met-35(O). 2 Using size exclusion chromatography and oxidation of A␤-(1-40) with H 2 O 2 , we have verified the attenuated fibril formation associated with A␤-(1-40)Met-35(O). However, the structural and conformational changes underlying this phenomenon have not yet been identified. Peptide fibril formation is a complex multistep reaction involving the transitional formation of numerous oligomeric (13) and protofibrillar (14,15) A␤ species. Clarification of what steps in the oligomerization cascade underlie the affected aggregation rate of A␤-(1-40)Met-35(O) would not only increase our understanding of the fibrillization reaction but also our understanding of the forces determining peptide structure and folding. This could in turn be important for understanding the potential toxicities of different A␤ species.
This study was aimed at investigating the consequences of Met-35 oxidation on the formation of small A␤ peptide oligomers and also at attempting to find oxidation-mediated ratelimiting steps in the fibrillization cascade of the Met-35(O)modified A␤-(1-40) peptide using a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer and a homebuilt electrospray device. Electrospray ionization (ESI) mass spectrometry has been used previously to detect and characterize A␤ peptides in vitro (16) as well as in vivo (17). Using gentle ESI conditions, small aggregates of A␤-  in water can be transferred to the gas phase and detected, making it possible to monitor the aggregation of A␤-(1-40) over time (16). The oligomer distribution detected by ESI mass spectrometry (16) is similar to that approached in the short irradiation time limit of photo-induced cross-linking experiments (18). An ad-vantage in using the high resolving power of a 9.4 T FTICR instrument is that homo-and heterooligomers of A␤-(1-40) and A␤-(1-40)Met-35(O) can be resolved. Furthermore, the sensitivity of the technique allows aggregation kinetics to be studied using nano-and micromolar concentrations of A␤, more closely resembling the in vivo situation (19).  was obtained from Bachem (Bachem AG, Bubendorf, Switzerland). A 4.0 M A␤-(1-40) solution was prepared in deionized water/acetic acid 99:1 (v/v), pH ϳ3. Immediately after dissolving the peptide, an aliquot was taken out, and 30% (w/w) hydrogen peroxide in water (Merck KGaA) was added to a final concentration of 2.7% (w/w). The experiment was also reproduced using H 2 O and 2.7% H 2 O 2 solutions in absence of acetic acid. Additional 2.0 M solutions were prepared in 49.5:49.5:1 deionized water/acetonitrile/acetic acid solution (v/v/v) with and without the addition of hydrogen peroxide to a final concentration of 2.7% (w/w).

Sample Preparation and Oxidation of Met-35-A␤-
Mass Spectrometry-A homebuilt instrument controlled the direct infusion where helium gas at a pressure of 1.3 bars was used to push the sample through a 30-cm fused silica capillary with an inner diameter of 25 m. One end of the capillary was lowered into the sample, and the other end, coated by a conductive graphite/polymer layer (20), was connected to ground, functioning as an electrospray needle. No sheath flow or nebulizing gas was used. The flow rate was ϳ40 nl/min. The ion source was coupled to an Analytica atmosphere-vacuum interface (Analytica, Branford, CT). A potential difference of 2.0 -3.0 kV was applied across a distance of 3-5 mm between the spraying needle and the inlet capillary. To confirm the formation of A␤-(1-40)Met-35(O), the peptide was sequenced by collision-induced dissociation (21) in the capillary/ skimmer region in the ESI atmosphere/vacuum interface by raising the capillary potential from ϳ60 to ϳ270 V. All mass spectra were acquired using a Bruker Daltonics (Billerica, MA) BioAPEX-94e superconducting, 9.4 T FTICR mass spectrometer (22) in broadband mode. Typically, 524,288 data points were acquired, adding a minimum of 128 spectra (ϳ3 min of acquisition time).

Analysis of A␤-(1-40) in H 2 O-
resulting from collision-induced dissociation in the capillary/ skimmer region. The b-ions covering residues 3-40 could be identified in this spectrum, b 1 ϩ and b 2 ϩ being outside the set m/z range. The mass difference between the most intense isotopic peak of b 34 4ϩ and the corresponding isotopic peak of b 35 4ϩ was found to be 147.0356 Da, within the experimental error of 1.5-2 ppm (24) from the theoretical monoisotopic mass of Met(O) (C 5 H 9 NO 2 S) of 147.0354 Da. As a comparison, the mass difference corresponding to a phenylalanine residue (C 9 H 9 NO) in this position would be 147.0684 Da. The almost complete sequence coverage demonstrates the ability to identify modifications of A␤ peptides while studying aggregation kinetics using the FTICR mass spectrometer. 3 U. Bartenstein, Bachem AG, personal communication.   (30).
From several structural predictions, Met-35 would be part of the second ␣-helix of the A␤-(1-40) molecule, comprising amino acid residues 28 -36 (31)(32)(33). Reportedly, A␤-(1-40) shows predominant ␣-helical structure at conditions that are thought to mimic the membrane environment, including SDS and trifluoroethanol (31,33,34). Previously, ␣-helix formation of A␤ was detected in water solution (35) but not known to be a prerequisite for ␤-sheet formation. However, recently careful temporal CD studies by Kirkitadze et al. (36) revealed that ␣-helical structure is formed in water solution by 18 biologically relevant forms of the A␤ peptide (including A␤-(1-40)) as a transitory structure en route to the ␤-sheet structure. Interestingly, Kallberg et al. (37) have identified the so-called ␣/␤ discordance as a predictor of ␤-amyloid formation, including the A␤ peptide, which further strengthens the ␣-helix3␤-sheet transition hypothesis. A␤ is, like SDS, an amphipathic molecule, and evidence was recently presented that it forms micelle-like structures (38), previously predicted to comprise a step in the fibrillization process (39). These allegedly constitute centers for fibril nucleation (38). One can therefore envision that, similarly to its dissolution by SDS, A␤ forms ␣-helix-competent environments on its own accord, in micell-like aggregates, once it reaches the critical "micellar" concentration needed for this structural transition.
The amphipathic nature of A␤ stems from its relatively polar N-terminal and extremely hydrophobic C-terminal region. There is also the central hydrophobic core, composed of amino acids 17-21. These hydrophobic regions, and in particular the C-terminal residues, Ile-41 and Ala-42 of the A␤1-42 peptide (36), are critical determinants of the aggregation rate (40,41). The oxidation may also change the stability of the second ␣-helix. However, the ␣-helix structure of A␤ has been detected just prior to maximum ␤-sheet formation when preparations are rich in higher molecular weight aggregates (36). Although the effect of Met-35(O) may be substantial in these instances, it is not likely to provide an explanation of our observations given the low concentrations and short time frames used in this work and when low molecular weight oligomers have been the focus of attention. Therefore, the driving force for the association of these small oligomeric structures may be more dependent on hydrophobic interactions than on secondary structural driving forces. A␤ dimer methionine sulfoxide groups pointing outwards, as suggested by molecular modeling (30) and indirectly supported by our observations, would reduce the total hydrophobic surface available for further molecular association. Interestingly, Shen and Murphy (28) have observed faster fibril growth rates in acidic acetonitrile/water solution. The secondary structure of dimers and trimers remains to be determined using direct methods such as NMR, and the discussion on ␣-helical contents has to be considered in this context.
In the experiments presented here, the ionization efficiencies and charge state distributions of monomer and dimers did not differ significantly between A␤-(1-40) and A␤-(1-40)Met-35(O) and therefore could be expected to be similar for trimers and tetramers as well, which is also supported by the fact that trimer and tetramer signal intensities are comparable after the initial lag phase. One should be cautious not to draw conclusions from the measurements of single charge states as charge state distribution can (and does, at least to a small degree) change upon oxidation of Met-35. The alteration of ionization efficiency and charge state distribution are possible sources of artifacts in electrospray ionization and could be caused by slight shifts in pH or altered potentials in the atmosphere/ vacuum interface due to contamination. Finally, the results also demonstrate the applicability of electrospray ionization mass spectrometry to the study of the formation of small amyloid peptide aggregates and their kinetics in the nanomolar to micromolar range. Larger aggregates (decamers or larger) of A␤ peptides should be possible to detect in mass spectrometers of this type if present in micromolar concentrations (22), provided they survive the transition from liquid to gas phase and provided that stable electrospray conditions can be obtained in such solutions.
In conclusion, we believe these results emphasize the need for more detailed molecular and mechanistic knowledge of the complex multistep A␤ fibrillization reaction to rapidly provide a more profound understanding of both cause and effect of A␤-mediated actions in vitro and also in vivo. In addition, the post-translational Met-35 modification, if it is effectuated in vivo, provides an intriguing possibility for the brain involving rerouting A␤ from a potential protofibrillar/fibrillar pathway to a pathway that opens up better prospects of keeping low molecular weight A␤ species in solution longer, thereby increasing the likelihood of proteolysis and clearance while decreasing the risks of deposition. The growing scientific foundation for a role of oxidative stress in AD brains (42) along with the recent shift in focus from the role of fibrillar to prefibrillar (13) and protofibrillar (43) A␤ species in the pathogenesis of AD make these observations particularly intriguing.