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N{O}REAPDOR Solid-State NMR and Ultra-High Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

A combined approach, using Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) and solid-state NMR (Nuclear Magnetic Resonance), shows a high degree of polymorphism exhibited by Aβ species in forming hydrogen-bonded networks. Two Alzheimer’s Aβ peptides, Ac-Aβ16−22-NH2 and Aβ11−25, selectively labeled with O and N at specific amino acid residues were investigated. The total amount of peptides labeled with O as measured by FTICR-MS enabled the interpretation of dephasing observed in N{O}REAPDOR solid-state NMR experiments. Specifically, about one-third of the Aβ peptides were found to be involved in the formation of a specific >C17O···H−15N hydrogen bond with their neighbor peptide molecules, and we hypothesize that the rest of the molecules undergo ± n off-registry shifts in their hydrogen bonding networks. S stability and folding in biological systems is strongly dependent on a network of hydrogen bonds. Building on fundamental insight into amyloid fibril structure provided by other solid-state NMR methods, N{O}REAPDOR solidstate NMR has been recently employed in probing >CO···H− N hydrogen bonding in amyloid fibrils formed by selectively O, N labeled Alzheimer’s amyloid-β (Aβ) peptides. To use this method, O enrichment is essential, because of the low natural abundance (0.04%) of the only magnetic isotope of oxygen. Simulations of N{O}REAPDOR dephasing data include the O enrichment level as an important parameter in the evaluation of the heteronuclear N−O dipole−dipole coupling constants, from which N···O internuclear distances and, therefore, the lengths of hydrogen bonds are calculated. However, elucidation of the precise degree of the O enrichment in peptides from either conventional mass spectrometry (MS) or O NMR spectroscopy is a challenge, because of the naturally abundant C (1.07%) isotope and an unknown degree of O and O enrichment in reagents used in peptide synthesis, all leading to a severe overlap in MS patterns from different isotopologues (CO, CO, etc.). O enrichment of amino acids is usually achieved by the use of O enriched water. Then, amino acids are Fmoc protected and used as the input reagent in the fast Fmoc peptide synthesis. Although the initial O and O enrichment in water could be determined with a high precision, O enrichment levels may change during the whole synthetic pathway. Moreover, for the samples of interest in this research, the important value is the O enrichment of one specific oxygen atom within an otherwise normal peptide. Most methods one could consider to accurately determine the O/O enrichment values would require chemistry such as acid hydrolysis or enzymatic digestion to cleave the peptide into individual amino acids and afterward measure the O enrichment of the individual amino acid. However, such strategies run a strong risk of scrambling the O labeling position and thus invalidating the measured O enrichment information, and such methods generally will also dilute the signal by inclusion of the oxygen atoms from other unlabeled amino acids. In a mass spectrum, the well resolved isotopic peaks of one molecule representing each isotopologue are called the isotopic fine structure of this molecule. In principle, the fine structure could be separated if the resolution is sufficiently high (typically requiring a resolving power m/Δm50% of 1−5 M at m/z 1000 depending on the elemental composition), but it remains a challenging task for most modern mass spectrometers. Ultrahigh resolution isotopic fine structure separations have to date been achieved using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) on molecules smaller than 1500 Da and are particularly focused on the determination of the elemental composition. The two Aβ samples analyzed in this study are Ac-Aβ16−22NH2 (Ac-KLV18( O)FF20( N)AE-NH2, abbreviated as Aβ16−22 hereafter) and Aβ11−25 (EVHHQKLV18( O)FFA21(U−C,N)EDVG), i.e., the same samples as previously studied by 15N{17O}REAPDOR. Here, using FTICR-MS, the total ratio of O enrichment in the two samples is determined. Since N is labeled (>98%) on Phe20 for Aβ16−22 or on Ala21 for Aβ11−25, and similarly C is labeled (>99%) on Ala21 for Aβ11−25, the formulas of the two peptides are written as C45H67O10N8 N (Mw 894.498124 Da) and C78 C3H119O23N20 N (Mw 1757.885869 Da), respectively. In order to calculate the ratio of O labeling, the two peaks corresponding to C-substituted and O-substituted peptides in the A+1 cluster (second isotopic Received: October 7, 2015 Revised: March 4, 2016 Published: March 17, 2016 Rapid Report pubs.acs.org/biochemistry © 2016 American Chemical Society 2065 DOI: 10.1021/acs.biochem.5b01095 Biochemistry 2016, 55, 2065−2068 This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.