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.

Desorption electrospray ionization mass spectrometry imaging of latent fingerprints revealed by Oil Red O.

Mass spectrometry imaging (MSI) is increasingly used to produce chemical images of latent fingerprints. Yet, the actual benefits of MSI for real case studies have to be assessed for fingerprints previously processed by forensic techniques. Here, we have evaluated the compatibility of desorption electrospray ionization (DESI) with the fingerprint enhancement technique involving Oil Red O (ORO). METHODS: To optimize the ionization step independently from surface extraction, the ORO reagent and its mixture with model compounds (triolein and linoleic acid) were first studied in solution using high-resolution electrospray ionization tandem mass spectrometry (ESI-MS/MS). Then, DESI-MSI experiments were performed in both polarity modes for ORO-processed fingermarks deposited on pieces of paper used as porous substrates. RESULTS: ESI-MS of ORO reveals a complex mixture of azo dyes. Two main impurities detected beside the targeted species were characterized using MS/MS and then were usefully employed to produce DESI-MS images of fingermarks, decreasing the scanning rate to get sufficient ion abundance from natural fingerprints. ORO did not prevent chemical profiling, and one major added value of this pink dye was to produce MS images with contrast that cannot be obtained optically for some colored substrates. CONCLUSIONS: DESI-MS has demonstrated imaging compatibility with the application of ORO used to enhance latent fingerprints on paper and could also enable chemical profiling in natural fingermarks. In addition, MS images of ORO impurities were of higher quality than optical ones for fingerprints revealed on colored paper.

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.

Covalent Attachment and Detachment by Reactive DESI of Sequence-Coded Polymer Taggants.

The use of sequence-defined polymers is an interesting emerging solution for materials identification and traceability. Indeed, a very large amount of identification sequences can be created using a limited alphabet of coded monomers. However, in all reported studies, sequence-defined taggants are usually included in a host material by noncovalent adsorption or entrapment, which may lead to leakage, aggregation, or degradation. To avoid these problems, sequence-defined polymers are covalently attached in the present work to the mesh of model materials, namely acrylamide hydrogels. To do so, sequence-coded polyurethanes containing a disulfide linker and a terminal methacrylamide moiety are synthesized by stepwise solid-phase synthesis. These methacrylamide macromonomers are afterward copolymerized with acrylamide and bisacrylamide in order to achieve cross-linked hydrogels containing covalently-bound polyurethane taggants. It is shown herein that these taggants can be selectively detached from the hydrogel mesh by reactive desorption electrospray ionization. Using dithiothreitol the disulfide linker that links the taggant to the gel can be selectively cleaved. Ultimately, the released taggants can be decoded by tandem mass spectrometry.

Reactive Desorption Electrospray Ionization Mass Spectrometry To Determine Intrinsic Degradability of Poly(lactic-co-glycolic acid) Chains.

Because of its speed, sensitivity, and ability to scrutinize individual species, mass spectrometry (MS) has become an essential tool in analytical strategies aimed at studying the degradation behavior of polyesters. MS analyses can be performed prior to the degradation event for structural characterization of initial substrates or after it has occurred to measure the decreasing size of products as a function of time. Here, we show that MS can also be usefully employed during the degradation process by online monitoring the chain solvolysis induced by reactive desorption electrospray ionization (DESI). Cleavage of ester bonds in random copolymers of lactic acid (LA) and glycolic acid (GA) was achieved by electrospraying methanol-containing NaOH onto the substrates. Experimental conditions were optimized to generate methanolysis products of high abundance so that mass spectra can be conveniently processed using Kendrick-based approaches. The same reactive-DESI performance was demonstrated for two sample preparations, solvent casting for soluble samples or pressed pellets for highly crystalline substrates, permitting to compare polymers with LA/GA ratios ranging from 100/0 to 5/95. Analysis of sample fractions collected by size exclusion chromatography showed that methanolysis occurs independently of the original chain size, so data recorded for poly(LA-co-GA) (PLAGA) copolymers with the average molecular weight ranging from 10 to 180 kDa could be safely compared. The average mass of methanolysis products was observed to decrease linearly (R2 = 0.9900) as the GA content increases in PLAGA substrates, consistent with the susceptibility of ester bonds toward solvolysis being higher in GA than in LA. Because DESI only explores the surface of solids, these data do not reflect bulk degradability of the copolymers but, instead, their relative degradability at the molecular level. Based on a "reactive-DESI degradability scale" such as that established here for PLAGA, the proposed method offers interesting perspectives to qualify intrinsic degradability of different polyesters and evaluate their erosion susceptibility or to determine the degradability of those polymers known to degrade via erosion only.