Using ion mobility spectrometry to understand signal dilution during tandem mass spectrometry sequencing of digital polymers: Experimental evidence of intramolecular cyclization.

RATIONALE: Optimizing the structure of digital polymers is an efficient strategy to ensure their tandem mass spectrometry (MS/MS) readability. In block-truncated poly(phosphodiester)s, homolysis of C-ON bonds in long chains permits the release of smaller blocks amenable to sequencing. Yet the dissociation behavior of diradical blocks was observed to strongly depend on their charge state. METHODS: Polymers were ionized in negative mode electrospray and activated in-source so that blocks released as primary fragments can be investigated using ion mobility spectrometry (IMS) or sequenced in the post-IMS collision cell. Collision cross sections (CCS) were derived from arrival times using a calibration procedure developed for polyanions using the IMSCal software. A multistep protocol based on quantum methods and classical molecular dynamics was implemented for molecular modeling and calculation of theoretical CCS. RESULTS: Unlike their triply charged homologues, dissociation of diradical blocks at the 2- charge state produces additional fragments, with +1 m/z shift for those holding the nitroxide α-termination and -1 m/z for those containing the carbon-centered radical ω-end. These results suggest cyclization of these diradical species, followed by H• transfer on activated reopening of this cycle. This assumption was validated using IMS resolution of the cyclic/linear isomers and supported by molecular modeling. CONCLUSIONS: Combining IMS with molecular modeling provided new insights into how the charge state of digital blocks influences their dissociation. These results permit to define new guidelines to improve either ionization conditions or the structural design of these digital polymers for best MS/MS readability.

Using Nitroxides To Model the Ion Mobility Behavior of Nitroxide-Ended Oligomers: A Bottom-up Approach To Predict Mobility Separation.

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.

Conception and Evaluation of a Library of Cleavable Mass Tags for Digital Polymers Sequencing.

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.