Multiplatform High-Definition Ion Mobility Separations of the Largest Epimeric Peptides.

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has become a versatile tool to fractionate complex mixtures, distinguish structural isomers, and elucidate molecular geometries. Along with the whole MS field, IMS/MS advances to ever larger species. A topical proteomic problem is the discovery and characterization of d-amino acid-containing peptides (DAACPs) that are critical to neurotransmission and toxicology. Both linear IMS and FAIMS previously disentangled d/l epimers with up to ∼30 residues. In the first study using all three most powerful IMS methodologies─trapped IMS, cyclic IMS, and FAIMS─we demonstrate baseline resolution of the largest known d/l peptides (CHH from Homarus americanus with 72 residues) with a dynamic range up to 100. This expands FAIMS analyses of isomeric modified peptides, especially using hydrogen-rich buffers, to the ∼50-100 residue range of small proteins. The spectra for d and l are unprecedentedly strikingly similar except for a uniform shift of the separation parameter, indicating the conserved epimer-specific structural elements across multiple charge states and conformers. As the interepimer resolution tracks the average for smaller DAACPs, the IMS approaches could help search for yet larger DAACPs. The a priori method to calibrate cyclic (including multipass) IMS developed here may be broadly useful.

Total Chemical Synthesis of Glycosylated TREM2 Ectodomain.

Mutations in a microglia-associated gene TREM2 increase the risk of Alzheimer's disease. Currently, structural and functional studies of TREM2 mainly rely on recombinant TREM2 proteins expressed from mammalian cells. However, using this method, it is difficult to achieve site-specific labeling. Here, we present the total chemical synthesis of the 116 amino acid TREM2 ectodomain. Rigorous structural analysis ensured correct structural fold after refolding. Treating microglial cells with refolded synthetic TREM2 enhanced microglial phagocytosis, proliferation, and survival. We also prepared TREM2 constructs with defined glycosylation patterns and found that glycosylation at N79 is critical to the thermal stability of TREM2. This method will provide access to TREM2 constructs with site-specific labeling, such as fluorescent labeling, reactive chemical handles, and enrichment handles, to further advance our understanding of TREM2 in Alzheimer's disease.

Top-Down Ion Mobility Separations of Isomeric Proteoforms.

Continuing advances in proteomics highlight the ubiquity and biological importance of proteoforms─proteins with varied sequence, splicing, or distribution of post-translational modifications (PTMs). The preeminent example is histones, where the PTM pattern encodes the combinatorial language controlling the DNA transcription central to life. While the proteoforms with distinct PTM compositions are distinguishable by mass, the isomers with permuted PTMs commonly coexisting in cells generally require separation before mass-spectrometric (MS) analyses. That was accomplished on the bottom-up and middle-down levels using chromatography or ion mobility spectrometry (IMS), but proteolytic digestion obliterates the crucial PTM connectivity information. Here, we demonstrate baseline IMS resolution of intact isomeric proteoforms, specifically the acetylated H4 histones (11.3 kDa). The proteoforms with a single acetyl moiety on five alternative lysine residues (K5, K8, K12, K16, K20) known for distinct functionalities in vivo were constructed by two-step native chemical ligation and separated using trapped IMS at the resolving power up to 350 on the Bruker TIMS/ToF platform. Full resolution for several pairs was confirmed using binary mixtures and by unique fragments in tandem MS employing collision-induced dissociation. This novel capability for top-down proteoform characterization is poised to open major new avenues in proteomics and epigenetics.

Binding Sites of a Positron Emission Tomography Imaging Agent in Alzheimer's ß-Amyloid Fibrils Studied Using 19F Solid-State NMR.

Amyloid imaging by positron emission tomography (PET) is an important method for diagnosing neurodegenerative disorders such as Alzheimer's disease. Many 11C- and 18F-labeled PET tracers show varying binding capacities, specificities, and affinities for their target proteins. The structural basis of these variations is poorly understood. Here we employ 19F and 13C solid-state NMR to investigate the binding sites of a PET ligand, flutemetamol, to the 40-residue Alzheimer's ß-amyloid peptide (Aß40). Analytical high-performance liquid chromatography and 19F NMR spectra show that flutemetamol binds the current Aß40 fibril polymorph with a stoichiometry of one ligand per four to five peptides. Half of the ligands are tightly bound while the other half are loosely bound. 13C and 15N chemical shifts indicate that this Aß40 polymorph has an immobilized N-terminus, a non-ß-sheet His14, and a non-ß-sheet C-terminus. We measured the proximity of the ligand fluorine to peptide residues using 19F-13C and 19F-1H rotational-echo double-resonance (REDOR) experiments. The spectra show that three segments in the peptide, 12VHH14, 18VFF20, and 39VV40, lie the closest to the ligand. REDOR-constrained docking simulations indicate that these three segments form multiple binding sites, and the ligand orientations and positions at these sites are similar across different Aß polymorphs. Comparison of the flutemetamol-interacting residues in Aß40 with the small-molecule binding sites in other amyloid proteins suggest that conjugated aromatic compounds preferentially bind ß-sheet surface grooves lined by aromatic, polar, and charged residues. These motifs may explain the specificity of different PET tracers to different amyloid proteins.