Innovation in analytical methodologies is of high impact for biomedical and environmental applications. In this context, electrokinetic methods represent powerful tools allowing selective and ultrasensitive separations. They are easy to run, versatile, of high efficiency and require very small sample amounts. One way to improve separations is to develop new nanosupports that can be integrated within the capillaries or microchannel, so as to increase separation selectivity and to generate specific interactions with some analytes^[1]. Thereon, we aim at developing Peptide Nanotubes (PNTs) as new nanosupports, as their structure can offer high diversity and surface functionalization, they are biocompatible and stable in different media. Their cylindrical structure could be used as sieving tools to separate small compounds, amplifying possible applications. The synthesis of PNTs depends on the cyclic peptide (CP) monomers structure and concentration. It is therefore crucial to realize the physicochemical characterization of these CPs. Then, we developed a new capillary electrophoretic method coupled on line with mass spectrometry via an electrospray interface (CE-ESI-MS) to characterize the monomers, and the study of their possible aggregation.

Metodology

We synthesized 8 CPs (Table 1) of three internal diameters via 8, 10 and 12 D, L alternated amino acids by Fmoc-solid phase peptide synthesis technique^[2]. They were purified by HPLC and the purity was determined by the development of CE-ESI-MS methodology, which allowed identifying their corresponding molecular ions, isotopes and some adducts with NH₄⁺ and Na⁺.

CE analysis were performed in zone mode (λ=200nm) at 25°C, with separation at 20kV along pressure application (50mbar). BGE used was 10mM ammonium carbonate (pH8.0)/EtOH (50/50, v/v), and 10mM ammonium formiate (pH3.0)/EtOH (50/50, v/v). The neutral marker was a mixture of dimethylformamide (UV detection) and glucose (MS detection). Fused silica capillaries were of  75µm internal diameter, 80cm total length and 20cm detection length. They were previously activated by successive flushes of NaOH 1M, NaOH 0.1M, H₂O and BGE (934mbar) during 15 min each. Sample concentration was 3mM (dissolved in 0.1mM BGE). MS measurements (SCAN and SIM) were done with a sheath liquid composed of H₂O/EtOH (10/90, v/v) containing 2.0% AcOH (0.6µL/min), a nebulizing gas (N_2,g) at 10psi pressure, and a drying gas (N_2,g) at 250°C (6L/min). Fragmentor voltage was fixed at 70V, and acquisition was performed in positive mode.

Results

The yields of synthesized CPs were between 42.0-90.0%, which are higher values than literature^[3]. These compounds are soluble in basic media (pH8.0), and dissolved in acidic media (pH3.0), a protonation is expected as well as an aggregation of monomers into PNTs. 

It was shown that their electrophoretic mobility (µ_ep) was in accordance with their global charge and mass, but unexpected behaviors could be due to specific cyclic configurations of peptides, due to their sequence nature. Furthermore, MS evidenced the absence of corresponding linear, protected/partially deprotected peptides, and assemblies, in the analysis conditions, proving their purity.

The effect of dissolution media on the µ_ep (calculated according to^[4]) was studied for CP-8 aaSerie3. This dissolved in basic conditions presents a µ_ep of -4.31±0.11x10^-5cm².V^-1.s^-1, in agreement with negatively charged compounds. When modifying analysis conditions to acidic media, a shift in mobility was observed, leading 1.11±0.02x10^-5cm².V^-1.s^-1, indicating a protonation of monomers and possible self-assembly. While, CP dissolved in acidic conditions, the µ_ep  is positive (2.35±0.01x10^-5cm²V^-1s^-1). When modifying analysis conditions to basic media, a negative value was determined (-2.94±0.3x10^-5cm²V^-1s^-1)^. These results seem to indicate a self-assembly between CPs in acidic media, with different stages, as µ_ep values are different according to pH modification pathway. To further in the interpretation, the MS signal of CP dissolved in acidic media was analyzed, showing a m/z value corresponding to twice molecular mass of monomer. This indicates a minima dimerization of CPs, and probably formation of PNTs.

Conclusions

We designed and synthesized eight new CPs that could provide PNTs of various properties. In view of aggregation as PNTs, their structure and purity had to be verified. Therefore, a new methodology based on CE-ESI-MS was set up. After optimizing various parameters, this methodology was demonstrated to be an innovative tool for easy and quick characterization of CP. When dissolved in acidic media, µ_ep were drastically modified possibly due to aggregation phenomenon. These pure CPs will thus be interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or pseudo-sieving tools for separative purposes.

 

[1]               M. Girardot, et al., Anal.Biochem, 435, 150, (2013).

[2]               W. C. Chan, P. D. White, Fmoc solid phase peptide synthesis, (2000).

[3]               S. F. Brady, et al., JOC, 44, 3101, (1979)

[4]               A. Varenne, et. al., Anal.Biochem., 315, 152 (2003).