Structural Characterization of Monomers and Oligomers of D-Amino Acid-Containing Peptides Using T-Wave Ion Mobility Mass Spectrometry
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The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer’s disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers.
KeywordsIon mobility mass spectrometry IM-MS D-amino acid containing peptides DAACPs Native state Organic solvent Monomer Dimer Oligomer Oligomerization pattern Metal adducts Collision cross-section CCS Conformational differences
D-amino acid containing peptides (DAACPs) are diastereomers with significant biological functions. Every amino acid (except glycine) can occur in two isomeric forms because of their ability to form stereoisomers around the central α-carbon atom. The DAACP and its corresponding L-form isomer constitute peptide epimers. Before the first DAACP, dermorphin, was isolated in 1981 , it was believed that all proteins and peptides were comprised of only L-amino acids. To date, more than 30 endogenous DAACPs have been found in living organisms , including crustaceans [3, 4], snails [5, 6, 7], spiders , frogs [9, 10, 11], and even mammals [12, 13]. For example, the spider-venom agatoxins , a crustacean hormone , and sheep crystallin proteins , all contain DAACPs. The D-amino acid residues produced by post-translational isomerization have been proven to be crucial to the biological activities of DAACPs. For example, the DAACP cardioactive peptide (NdWFamide) significantly enhances the beating amplitude of an Aplysia heart at nM level, whereas the L-isomer has little effect even at μM level [7, 17]. Achatin I, which contains D-phenylalanine, can excite muscles of the snail, but its L-isomer does not have this function . These observations and reports raise a series of intriguing questions. For example, why are the activities of D/L isomers so different? How much do D/L isomers differ in conformation? Which factors may enhance the differences of these isomers? The first step towards answering these intriguing questions is to develop methods that characterize these structural isomers.
D/L isomer separation is challenging for mass spectrometry (MS) as the isomerization does not change the elemental composition, molecular formula, or mass of the peptide. However, the gas-phase basicities of the diastereomers can be differentiated when they break apart into fragments  and thus can be manifested as different branching ratios among product ions . As a result, MS has become a powerful tool to study DAACPs [20, 21, 22]. Fragmentation patterns generated during MS/MS sequencing were used to probe the thermochemical difference between peptide diastereomers during collision-induced dissociation (CID) , electron capture dissociation , or radical-directed dissociation (RDD) . Although excellent differentiation and quantitation between D/L peptide isomers can be accomplished by these strategies, localization of D-amino acid in peptides is still difficult, as measurement of fragment ion intensities cannot provide accurate positional information of D-amino acids. Our previous study introduced a novel ion mobility mass spectrometry (IM-MS) based strategy enabling site-specific characterization of DAACP isomers to localize D-amino acids .
The folding of peptide monomers and oligomers, including conformational changes and oligomerization patterns, have been investigated by IM-MS [24, 25, 26, 27]. In neurodegenerative diseases, amyloid cascades transform the native unstructured peptide into ß-sheet oligomers forming insoluble plaques [28, 29, 30]. The Bowers group studied this conformational conversion with IM-MS. Their study revealed that the unstructured soluble peptide assemblies and insoluble amyloid plaque followed different oligomerization patterns, as shown by different distribution trends of collision cross-section as a function of aggregation state . Although neuropeptides and neurotransmitters are typically present at very low concentrations throughout the nervous system, their concentrations could be much higher in neuronal organelle. For example, the concentration of a neuropeptide in a large dense-core vesicle is 3–10 mM , the concentration of acetylcholine (ACh) in synaptic vesicles is ~260 mM , the vesicular concentration of catecholamine in chromaffin cells is 190–300 mM , and that of dopamine in midbrain neurons is ~300 mM . This paradox prompted an interesting question whether concentrated DAACPs could also form oligomers and, if so, what kind of biomedical consequences the DAACP oligomerization might have. Elucidating the oligomerization pattern and structural changes is important for understanding the activities of DAACPs. Therefore, we set out to study the conformational differences of monomer, dimers, and oligomers, using T-Wave IM-MS.
Peptide oligomers that form different assemblies in solution have been characterized [35, 36, 37]. The growth of the oligomers was monitored with IM-MS by measuring collision cross-section (CCS). The correlation function between CCS (Y) and oligomer size (n) is different depending on the assembly types . For example, in spatially isotropic self-assembly, the relation is Y = Ymon * n2/3, where Ymon is the monomer CCS; whereas in fibrillary self-assembly, Y = a * n + k, where a and k are constants. Thus, the types of self-assembly could be distinguished via the correlation function between CCS and oligomer size, referred to as oligomerization pattern hereafter. In this study, we investigated the oligomerization patterns and conformational changes induced by metal binding and other factors. According to our study, dimerization, oligomerization, and metal adduct formation augment the structural differences of D/L peptide isomers and thus improving the separation and resolution of these important epimers in IM-MS.
Chemicals and Biological Samples
Methanol was obtained from Sigma-Aldrich (St. Louis, MO, USA). Optima grade water, acetonitrile (ACN), acetic acid (HAc), and ammonium acetate (certified ACS) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Peptide standards were purchased from American Peptide Company. [D-Ala]-dermorphin (Y(D/L)AFGYPS), [D-Phe]-achatin-I (G(D/L)FAD), and their all-L forms were synthesized in Biotechnology Center, University of Wisconsin-Madison.
CCS Calibration and Measurement
Polyalanine was suspended in 50% ACN, 1% HAc at 0.1 mg mL–1, and acquired under six different T-Wave IM wave velocity (WV)/wave height (WH) ratios (m s–1 V–1): 500/30, 500/35, 600/35, 600/40, 700/35, and 800/40. WH refers to the magnitude of the sequential voltage pulses that make up the traveling wave, and WV refers to the propagation speed of those pulses. Instrument parameters were optimized to minimize ion heating during transmission. These parameters include the spray voltage (nano ESI capillary voltage), cone voltage, extraction cone voltage (orthogonal skimmer cone), trap bias (energy for injection into helium cell), and helium cell DC (energy for injection into IM cell) set to 2.0 kV, 30 V, 4 V, 35 V, and 15 V, respectively. The source temperature was set to 70 oC. Data processing was conducted using MassLynx 4.1 and DriftScope 2.1. The measured drift time profiles were calibrated into absolute CCS with a home-built software package.
IM-MS was performed with the nanospray source of the Synapt G2 HDMS (Waters, Milford, MA, USA) in positive ionization mode with nitrogen as IM-MS buffer gas. The average pressures (mbar) of the instrument backing, source, trap, and helium cell were 2.73, 1.22 × 10–3, 2.66 × 10–2, and 1.46 × 103, respectively. For experiments conducted with aqueous buffer, analytes were dissolved in 500 mM ammonium acetate buffer at concentrations of 1 mg/ml (~1.3 mM). For experiments conducted in organic solvent, analytes were dissolved in 50:50 water/methanol solution, and sample concentration was 1 mg/ml (~1.3 mM). The samples were directly infused into a Synapt G2 HDMS mass spectrometer equipped with a nano-ESI ion source at a rate of 0.05 μL/min. The ion of interest was isolated by the quadrupole. The CCS distributions were converted from extracted drift time distributions with a window of 0.001 Th.
Results and Discussion
Dimerization Enhances Structural Difference in Both Aqueous and 50% Methanol/water Solvents
Although the structures of diastereomers are very different around the chiral center and in most cases could be easily separated by chiral or even normal reversed-phase HPLC, the chirality did not much affect the collision cross-section or the mobility of the molecules as was measured in IM-MS. The reason might be for short peptides (e.g., 4–7 amino acid peptide), their secondary structures were not significantly affected by isomerization, especially when measured in the gas phase. The relatively low resolution of IM-MS, or T-Wave IM-MS more specifically, could further contribute to the small differences observed. IM-MS separates gas-phase ions based on their differential mobility through a buffer gas. IM-MS gains advantages on rapid separation speed (on the millisecond timescale versus HPLC typically on the scale of seconds), and low detection limit (attomole amounts, which is a 10-fold improvement compared with HPLC) . However, T-Wave IM-MS does suffer from low resolving power (R < 100 Ω/ΔΩ), meaning that an ion with Ω of 1000 Å2 can theoretically be well separated from an ion with ±10 Å2 difference in Ω . As a result, although the local conformation around the chiral center of the DAACP monomers differs largely, the conformational difference we observed in IM-MS could be quite small. A benchmark study performed by the Ruotolo’s group showed the relative standard deviation values in CCS, as a function of Weight Height, for alcohol dehydrogenase (ADH) tetramers demonstrating a small amount of variation, ranging from 0.1% to 0.3% . The relative CCS differences of the dimers observed in our study, being 1% to 2.9%, were 10 to 30 times larger than the typical systematic variation, suggesting that the conformational differences between the D/L dimers measured in IM-MS study are rather significant. Although the measured peptides may have different conformations in organic or native solution, the overall trend in both native (aqueous) and denatured conditions suggested that dimerization enhanced conformational differences between D/L peptide isomers.
Oligomers Were Detected for Dermorphin Isomers in Aqueous and 50% Methanol/water Solvents with Unique Conformers to D- or L-isomers Being Observed
Most of the observed oligomers formed by L-form dermorphin are present in a more extended conformation compared with their D-form counterparts. For all the L-form oligomers, we compared the conformations with those of D-form counterparts by calculating the CCS difference between L- and D-form oligomers under six different sets of wave height/wave velocity measurements. The results were shown in Figure 5b, and the CCS difference was increased from null for the monomer to 1~4 Å2 for different oligomers. L-form oligomers exhibited larger CCS, suggesting that the L-form oligomers have more extended conformation in comparison to the D-forms. It might be very possible that the trend was solvent-specific. For the limited IM-MS oligomer peaks detected in aqueous experiments, the D-forms exhibited slightly more extended conformation compared with L-form oligomers; however, the differences are relatively modest. Overall, our observation supported that the chiral center played an important role in forming oligomers and ion mobility was capable of detecting those small changes.
Other techniques, such as native gel analyses, high-resolution atomic force microscopy, electron microscopy, and molecular structural modeling could be combined with IM-MS to verify peptide oligomerization in vitro. Applying native gel analyses of full-length tau and deletion constructs, the Bowers’ group demonstrated that the N-terminal region produced multiple bands, which was consistent with oligomerization . High resolution atomic force microscopy was used to directly image populations of small oligomers and observe features that can be attributed to oligomers with different number of subunits . By combining electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods, Schmidt and co-workers determined the molecular architecture of a fibril formed by Aβ(1–42). Their model revealed that the individual layers of the Aβ fibril were formed by peptide dimers with face-to-face packing . To confirm the oligomer formation of DAACPs and their oligomerization pattern in large dense core vesicles, we are going to employ electron microscopy, high resolution atomic force microscopy, and molecular simulations to further validate our hypothesis proposed by the IM-MS study. To date, we have tested several other DAACPs, including G(D/L)FFD and Y(D)RFG, demonstrating potential applicability of the methods to other D/L pairs. Additional DAACP isomers will be investigated to reveal general trends of DAACP oligomerization.
Potassium Adducts of Dermorphin Isomers
We studied dimerization, oligomerization, and metalation of the D/L peptide isomers using IM-MS. Dimers, oligomers, and metal adducts were detected in both aqueous and 50% methanol/water solutions. The relative CCS difference of the peptide isomers increased when monomers were converted into dimers, indicating that dimerization enhanced the structural difference of peptide isomers in both aqueous and 50% methanol/water solutions. In both solvent systems, oligomers were detected. The IM-MS results showed some conformers found only for D- or L-isomers, suggesting the importance of chiral center in the oligomerization process. The CCS versus aggregation state distribution of the dermorphin isomers exhibited a similar trend, whereas oligomerization enlarged the CCS difference from null for the monomer to 1~4 Å2 for different oligomers. Potassium adducts augmented the structural differences between D/L isomers, and the dynamic conversion of oligomer conformations showed 50/50 mixture presented unique conformations in either D- or L-forms. In summary, we demonstrate that IM-MS can be utilized to probe the structural differences of D/L peptide isomers at dimer, oligomer, and metalation. These differences may provide helpful molecular clues to address the intriguing question why D/L isomers differ significantly in biological activity. Further biochemical investigation and in-depth characterization of structure–function relationship of DAACPs in monomer and oligomer conditions will be needed to gain a clear mechanistic understanding of the process.
The authors thank Dr. Matthew Glover in the Li Research Group for critical reading of the manuscript and helpful discussions. This work is supported in part by the National Science Foundation grant (CHE-1413596) and the National Institutes of Health grants (1R01DK071801 and 1R56DK071801). L.L. acknowledges a Vilas Distinguished Achievement Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin-Madison School of Pharmacy.
- 3.Soyez, D., Van Herp, F., Rossier, J., Le Caer, J.P., Tensen, C.P., Lafont, R.: Evidence for a conformational polymorphism of invertebrate neurohormones. D-amino acid residue in crustacean hyperglycemic peptides. J. Biol. Chem. 269, 18295–18298 (1994)Google Scholar
- 5.Kamatani, Y., Minakata, H., Kenny, P.T., Iwashita, T., Watanabe, K., Funase, K., Sun, X.P., Yongsiri, A., Kim, K.H., Novales-Li, P., Novales, T.N., Lanapi, C.G., Takeuchi, H., Nomoto, K.: Achatin-I, an endogenous neuroexcitatory tetrapeptide from Achatina fulica Ferussac containing a D-amino acid residue. Biochem. Biophys. Res. Commun. 160, 1015–1020 (1989)CrossRefGoogle Scholar
- 15.Soyez, D., Vanherp, F., Rossier, J., Lecaer, J.P., Tensen, C.P., Lafont, R.: Evidence for a conformational polymorphism of invertebrate neurohormones—D-amino-acid residue in crustacean hyperglycemic peptides. J. Biol. Chem. 269, 18295–18298 (1994)Google Scholar
- 26.Bernstein, S.L., Dupuis, N.F., Lazo, N.D., Wyttenbach, T., Condron, M.M., Bitan, G., Teplow, D.B., Shea, J.E., Ruotolo, B.T., Robinson, C.V., Bowers, M.T.: Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease. Nat. Chem. 1, 326–331 (2009)CrossRefGoogle Scholar
- 31.Hrdina, P.D., Siegel G.J., Agranoff B.W., Albers R.W., Fisher S.K., Uhler M.D.: Basic neurochemistry: molecular, cellular, and medical aspects, 6th edition. Lippincott-Raven: Philadelphia (1999) Available at: http://www.ncbi.nlm.nih.gov/books/NBK20385/. Accessed 21 June 2016
- 33.Wightman, R.M., Jankowski, J.A., Kennedy, R.T., Kawagoe, K.T., Schroeder, T.J., Leszczyszyn, D.J., Near, J.A., Diliberto Jr., E.J., Viveros, O.H.: Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. U. S. A. 88, 10754–10758 (1991)CrossRefGoogle Scholar
- 34.Pothos, E.N., Davila, V., Sulzer, D.: Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size. J. Neurosci. 18, 4106–4118 (1998)Google Scholar
- 35.Sawaya, M.R., Sambashivan, S., Nelson, R., Ivanova, M.I., Sievers, S.A., Apostol, M.I., Thompson, M.J., Balbirnie, M., Wiltzius, J.J., McFarlane, H.T., Madsen, A.O., Riekel, C., Eisenberg, D.: Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447, 453–457 (2007)CrossRefGoogle Scholar
- 43.Feinstein, H.E., Benbow, S.J., LaPointe, N.E., Patel, N., Ramachandran, S., Do, T.D., Gaylord, M.R., Huskey, N.E., Dressler, N., Korff, M., Quon, B., Cantrell, K.L., Bowers, M.T., Lal, R., Feinstein, S.C.: Oligomerization of the microtubule-associated protein tau is mediated by its N-terminal sequences: implications for normal and pathological tau action. J. Neurochem. 137, 939–954 (2016)CrossRefGoogle Scholar