RESUMEN
Block-truncated poly(phosphodiester)s are digital macromolecules storing binary information that can be decoded by MS/MS sequencing of individual blocks released as primary fragments of the entire polymer. As such, they are ideal species for the serial sequencing methodology enabled by MS-(CID)-IMS-(CID)-MS coupling, where two activation stages are combined in-line with ion mobility spectrometry (IMS) separation. Yet, implementation of this coupling still requires efforts to achieve IMS resolution of inner blocks, that can be considered as small oligomers with α termination composed of one nitroxide decorated with a different tag. As shown by molecular dynamics simulation, these oligomers adopt a conformation where the tag points out of the coil formed by the chain. Accordingly, the sole nitroxide termination was investigated here as a model to reduce the cost of calculation aimed at predicting the shift of collision cross-section (CCS) induced by new tag candidates and extrapolate this effect to nitroxide-terminated oligomers. A library of 10 nitroxides and 7 oligomers was used to validate our calculation methods by comparison with experimental IMS data as well as our working assumption. Based on conformation predicted by theoretical calculation, three new tag candidates could be proposed to achieve the +40 Å2 CCS shift required to ensure IMS separation of oligomers regardless of their coded sequence.
RESUMEN
A library of phosphoramidite monomers containing a main-chain cleavable alkoxyamine and a side-chain substituent of variable molar mass (i.e. mass tag) was prepared in this work. These monomers can be used in automated solid-phase phosphoramidite chemistry and therefore incorporated periodically as spacers inside digitally-encoded poly(phosphodiester) chains. Consequently, the formed polymers contain tagged cleavable sites that guide their fragmentation in mass spectrometry sequencing and enhance their digital readability. The spacers were all prepared via a seven steps synthetic procedure. They were afterwards tested for the synthesis and sequencing of model digital polymers. Uniform digitally-encoded polymers were obtained as major species in all cases, even though some minor defects were sometimes detected. Furthermore, the polymers were decoded in pseudo-MS3 conditions, thus confirming the reliability and versatility of the spacers library.
