# Combining Laser-Induced Edition to Collision-Induced Sequencing of Digital Oligomers in a Single Gas-Phase Experiment

Clothilde Comby-zerbino, Jean-françois Lutz, Luke Macaleese, Laurence Charles

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Clothilde Comby-zerbino, Jean-françois Lutz, Luke Macaleese, Laurence Charles. Combining Laser-Induced Edition to Collision-Induced Sequencing of Digital Oligomers in a Single Gas-Phase Experiment. Rapid Communications in Mass Spectrometry, 2025, 10.1002/rcm.10016. hal-05394198

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RESEARCH ARTICLE

OPEN ACCESS

# Combining Laser-Induced Edition to Collision-Induced Sequencing of Digital Oligomers in a Single Gas-Phase Experiment

Clothilde Comby-Zerbino<fnref n="1" /> | Jean-François Lutz² | Luke MacAleese<fnref n="1" /> | Laurence Charles³

<figure><img src="image_3.png" /></figure>

**Received:** 19 December 2024 | **Revised:** 12 February 2025 | **Accepted:** 18 February 2025

**Funding:** The authors received no specific funding for this work.

**Keywords:** digital polymers | laser-induced dissociation | MS/MS sequencing | secure communications

## ABSTRACT

**Rationale:** To increase the confidentiality level of molecular tags, binary information can be hidden in sequence-defined oligomers using isobaric monomers including a photo-sensitive one, which mass can be modified upon light irradiation. Reading such secured data requires light edition prior to MS/MS sequencing, which was investigated here in a single gas-phase workflow.

**Methods:** Oligo (phosphodiester)s made of units with either light-sensitive *ortho*- or light-inert *para*-nitrobenzyl side-group were electrosprayed as deprotonated oligomers and sampled in an ion trap instrument equipped with various light sources. Nanosecond-pulsed laser (266 or 365 nm) and CW laser (375 nm) irradiations were compared for their efficiency to induce photo-cleavage of all *o*-nitrobenzyl moieties in laser-induced dissociation and to produce sufficient amounts of edited oligomers to achieve full coverage of their sequence in collision-induced dissociation.

**Results:** While cleavage of photo-sensitive moieties was never fully achieved with the pulsed laser, photo-cleavage of all *o*-nitrobenzyl side-groups readily occurred with the CW laser, as monitored by successive losses of *o*-nitrobenzaldehyde from the irradiated chains. Time required for maximal production of edited oligomers increased with their number of reactive units but shorter irradiation steps were found sufficient to produce enough secondary precursors to achieve full sequence coverage in MS/MS.

**Conclusions:** Laser-induced modification of light-sensitive units allowed monotonic sequences of two isobaric monomers to be turned into MS/MS readable sequences. This gas-phase experiment enables a much higher throughput compared with the 1-h irradiation requested for the oligomer edition when performed in solution.

## 1 | Introduction

Digital polymers are synthetic macromolecules designed to store binary information at the molecular level [1]. They are commonly prepared with co-monomers of different mass defined as the letters of an alphabet and arranged in a controlled sequence to write messages. From their early development, these new synthetic polymers have been foreseen as tags for

anti-counterfeiting purposes [2]. This is because such an application does not rely on long macromolecules of high storage capacity but, instead, on small coded oligomers that are less demanding in terms of synthesis efforts. Most importantly, short chains with typically less than 20 units can have their sequence fully covered by tandem mass spectrometry (MS/MS), a mandatory condition for error-free reading of such coded information. Using collision-induced dissociation (CID), reliable decoding

<footnote n="1">¹Université Claude Bernard Lyon 1, CNRS, Institut Lumière Matière (iLM), Lyon, France | ²Université de Strasbourg, CNRS, Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Strasbourg, France | ³Aix Marseille Université, CNRS, Institut de Chimie Radiculaire (ICR), Marseille, France</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2025 The Author(s). *Rapid Communications in Mass Spectrometry* published by John Wiley & Sons Ltd.</footnote>
has been reported for a variety of sequence-defined oligomers such as poly(alkoxyamine amide)s [3], poly(triazole amide)s [4], polyurethanes [5], poly(alkoxyamine phosphodiester)s [6], poly(ester amide)s [7], poly(urethane amide)s [8], poly(succinimide thioester)s [9, 10], poly(N-substituted urethane)s [11], polyesters [12], and poly(aminophosphodiester)s [13].

Practical use of sequence-defined oligomers as taggants has first been demonstrated for oligourethanes [5], now commercially available under the trademark name Poltag®. They have been incorporated in a variety of materials such as 3D-printed photocured methacrylate resins [5], acrylate-based intraocular implants [14], and model in vivo implants [15], then extracted by sonicating small pieces of tagged materials in small volumes of solvent further subjected to electrospray ionization (ESI). Alternatively, they were covalently attached to the mesh of model materials and detached by reactive desorption electrospray ionization (DESI), using dithiothreitol to selectively cleave the disulfide bond that links the taggant to the gel [16]. For best MS/MS sequencing, the negative ionization mode was used to generate gas-phase oligourethanes deprotonated at one end-group, which led to easy-to-read CID spectra with a single series of product ions spaced by the mass of one or the other coded monomer [17]. Although the simplicity of CID pattern is a clear advantage to ensure full sequence coverage of digital oligomers, it could be a real drawback when they are used for secure communications. Keeping MS/MS as a fast and accurate method to decode informational tags, increasing the security level of these communication media consists of adding a supplemental encryption dimension. This is most often done with multicomponent libraries containing oligomers with different chain-length, sequence, and mass to compose 2D barcodes that can be correctly reconstructed only by authorized readers aware of specific reading rules. These rules can be quite simple, for example, ordering coded sequences from the shortest to the longest as reported with oligourethane mixtures used to label different commodity polymers [18], or far more complex, as recently proposed by Zhang and coworkers with the reading order of dithiosuccinimide-based coded oligomers defined by their relative concentration as determined after deconvolution of size exclusion chromatography (SEC) traces [19].

Yet, in addition to high security level, practical applications of taggants to certify authenticity of products also require high throughput methods with minimal sample preparation and fast separation of mixtures. For example, the analytical workflow involving an

extraction step to recover oligourethane oligomers from tagged materials prior to their ESI-MS/MS characterization was advantageously accelerated using DESI for direct sampling of taggants that label materials at their surface [20]. In another application, an encryption strategy was conceived with the building blocks of sequence-defined poly(alkoxyamine phosphodiester)s prepared with a coding alkyl segment of the same mass but different architectures: accordingly, they could be distinguished within milliseconds using ion mobility spectrometry (IMS), allowing the digital sequence determined by MS/MS to be decrypted according to rules associated to IMS outputs [21]. A powerful strategy in terms of secure communications is to employ sequence-defined oligomers carrying hidden messages that can only be revealed using proper external stimulus such as light. This photo-editing concept has been successfully demonstrated with oligomers made of isobaric co-monomers containing either light-sensitive o-nitrobenzyl or light-inert p-nitrobenzyl pending group [22]. As illustrated in Figure 1, a chain composed of isobaric units is meaningless but contains a hidden message that can be revealed by MS/MS after irradiation at 365 nm. Using light as a trigger indeed permits to specifically cleave o-nitrobenzyl side-chains to produce new 154.0 Da units defined as bits-0 while unmodified p-nitrobenzyl monomers (289.0 Da) become bits-1.

However, the photo-edition step implemented in this latter work required 1-h irradiation of sample solutions under argon atmosphere in a closed box. Here, we have explored the capabilities of laser-induced dissociation (LID) to photo-edit these oligomers in the gas phase, on-line with their subsequent sequencing, as it is acknowledged that photochemical reactions can occur with high efficiency and at high rate upon laser irradiation of selected ions in vacuum [23].

## 2 | Experimental Section

### 2.1 | Chemicals

Methanol used to prepare polymer samples was from VWR International (Fontenay-sous-Bois, France). Synthesis of oligomers investigated in this study was made by automated phosphoramidite chemistry on an Expedite 8900 DNA synthesizer (Applied Biosystems, Waltham, MA) as previously described [22]. The oligomers were prepared on a thymidine-loaded controlled pore glass solid-support, cleaved, and purified on a reverse-phase cartridge. Their thymidine (T) end-group comes from the solid-support.

<figure><img src="image_4.png" /><figcaption>**FIGURE 1** | Hidden messages composed with isobaric units can only be revealed by MS/MS after photo-cleavage of the o-nitrobenzyl side group in light-sensitive monomers (pale grey) while light-inert p-nitrobenzyl monomers (dark grey) remain unchanged.</figcaption></figure>
## 2.2 | Mass Spectrometry

For all experiments, an LTQ Velos ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) was used to generate deprotonated oligomers in negative mode ESI from ~5 µM solutions in methanol. CID was performed in trap with collisional energy values reported in instrument specific normalized collisional energy (NCE) units. The NCE concept is specific to Thermo Fisher Scientific ion traps and accounts for the near-linear correlation between the collision energy required to achieve optimum fragmentation efficiency of an ion with its *m/z* value [24]. For LID experiments, MS² spectra were measured by irradiating mass selected precursor ions during the instrument-tunable ion activation time with collisional energy set to 0 NCE. Two laser sources were used in this study, either a UV-Vis optical parametric oscillator (Horizon OPO pumped at 10 Hz with the third harmonic of a Surelite I Nd:YAG laser Continuum, Amplitude Laser Inc., Santa Clara, CA) set at either 266 or 365 nm, or a continuous wave (CW) laser (Oxxius, Lannion, France) with fixed 375 nm wavelength. The pulse duration was 7 ns, and the energy per pulse was varied between 0.3 and 2.5 mJ by manual adjustment of the QSwitch delay on the OPO pump, while the CW laser power was varied from 0 to 110 mW via the software interface. The CW laser was fired upon reception of the ion activation trigger extracted from the MS: the irradiation time was strictly proportional to the applied MS activation time parameter, varied from 10 to 1500 ms. The number of OPO pulses was varied from 1 to 100 with a combination of a delay generator (DG645, Stanford Research Systems, Sunnyvale, CA), an Arduino Uno microcontroller for MS/laser synchronization logic and a mechanical shutter (SH05/TSC001, Thorlabs, Newton, NJ)

to allow the laser pulses only during ion activation time. MS³ experiments were performed by adding to the LID MS² step a secondary precursor ion selection and CID activation with collision energy varied up to 25 NCE.

# 3 | Results and Discussion

## 3.1 | Photo-Dissociation Mechanism and Nomenclature

LID-MS generally consists in irradiating mass selected ions with photons with appropriate wavelength so that their energy can be absorbed and lead to the precursor ion dissociation. Depending on the wavelength, but also on the irradiation conditions (peak power and duration), various energy accumulation/relaxation mechanisms may be at play and yield different types of fragment ions. In the UV range explored here, the absorption of photons tends to correspond to transitions toward electronically excited states of the ions. Absorption of UV photons can trigger isomerization phenomena that can be monitored by IMS [25–31] but also chemical reactions in the excited state, such as ion-molecule reactions [32] or intramolecular reactions such as photo-dissociation of *o*-nitrobenzyl groups investigated here. This reaction is expected to induce elimination of *o*-nitrobenzaldehyde (135 Da) from each light-sensitive *o*-nitrobenzyl moiety in the irradiated chains, as occurring in solution according a Norrish type II mechanism (Scheme 1) [33]. Progress of the reaction will hence typically be controlled by monitoring appearance of MS signals with –135 Da shifts from the precursor ion, the number of which should be equal to the number of *o*-nitrobenzyl units in the activated species.

<figure><img src="image_5.png" /><figcaption>**SCHEME 1** | Photo-elimination of *o*-nitrobenzaldehyde (135 Da) from *o*-nitrobenzyl side-group in photo-sensitive units of investigated oligomers.</figcaption></figure>
The general nomenclature adopted throughout this paper to describe investigated species is the following: nP<sub>i/j</sub>, with n the total number of units, j the original number of ortho-units and i the number of cleaved ortho-units after LID. Because all studied oligomers are octamers designed to store 8 bits (hence coding any character of the ASCII alphabet), n=8 is omitted for the sake of simplicity. Accordingly, as compared with P<sub>0/j</sub> starting oligomers, products formed upon LID are expected to be P<sub>1/j</sub> species, with the i value (i ≤ j) qualifying the reaction efficiency. For preliminary experiments, two octamers (2544.4 Da) were used: the P<sub>0/3</sub> analyte aimed to evaluate the efficiency of LID at producing the targeted P<sub>3/3</sub> species of mass 2149.3 Da and P<sub>0/0</sub> (i.e., composed of light-inert *p*-nitrobenzyl monomers only) as a control of the specificity of the LID reaction. Because their constituting *o*-nitrobenzyl and *p*-nitrobenzyl units are isobars (289.0 Da), the two starting oligomers exhibit the same MS profile: in the negative ion mode, they are preferentially detected as triply deprotonated molecules at *m/z* 850.4 (Figure S1, supporting information). Accordingly, all LID experiments described hereafter have been performed for precursor ions at the 3− charge state (i.e., P<sub>0/j</sub><sup>3−</sup>). The two oligomers also display the same CID pattern, which reveals a monotonic sequence (Figure S2, supporting information): these deprotonated species dissociate via cleavages of backbone bonds of the poly(phosphodiester) chains, independently of pending groups (Scheme S1, supporting information) [34].

## 3.2 | LID With a Pulsed Laser

The initial choice for a pulse laser emitting at 266 nm was motivated by (i) the presence of an intense band around the 250-nm absorption of nitrotoluene, (ii) the large availability of this wavelength (fourth harmonic of widespread Nd:YAG lasers),

and (iii) the increased possibility to explore high peak powers compared with other wavelengths, which generation by OPO may request up to two supplementary non-linear optics stages and resulting lower powers. The prominent [P<sub>0/3</sub> − 3H]<sup>3−</sup> ions were first isolated in the ion trap and irradiated (1 pulse) with a UV laser emitting at 266 nm. As depicted in Figure 2, two consecutive 135 Da losses are observed to occur from the *m/z* 850.4 precursor ion, yielding [P<sub>1/3</sub> − 3H]<sup>3−</sup> at *m/z* 805.4 and [P<sub>2/3</sub> − 3H]<sup>3−</sup> at *m/z* 760.4, respectively. The increase of the laser pulse energy does not allow a third *o*-nitrobenzaldehyde neutral to be eliminated and, counter-intuitively, the relative amounts of fragments P<sub>1/3</sub><sup>3−</sup> and P<sub>2/3</sub><sup>3−</sup> decrease to the point where P<sub>2/3</sub><sup>3−</sup> tends to zero towards 2 mJ/pulse (Figure S3, supporting information). Instead, the production of small singly charged fragments is favored (Figure 2A), such as deprotonated units observed at *m/z* 288.0 from the lowest laser pulse energy used in these experiments (0.3 mJ/pulse). As the pulse energy is further enhanced, additional low *m/z* ions appear (see structural assignments in Scheme S2, supporting information). Overall, experiments performed at 266 nm do not allow photo-cleavage of all *o*-nitrobenzyl groups while opening competing fragmentation routes that mainly yield internal fragments (hence useless for sequencing).

These undesired secondary fragmentations are also produced when irradiating the [P<sub>0/0</sub> − 3H]<sup>3−</sup> ions at 266 nm (Figure S4A, supporting information), which indicates their lack of specificity for the *o*-nitrobenzyl moieties. However, they are not observed when irradiation is performed at 365 nm, regardless of the number of *o*-nitrobenzyl units (Figure 2B and Figure S4B, supporting information). Thus, 365 nm represents an interesting alternative wavelength, which best mimics irradiation conditions when performed in solution and avoids undesired fragmentation. The absence of these small *m/z* fragments confirms that their formation

<figure><img src="image_6.png" /><figcaption>**FIGURE 2** | LID spectra recorded upon irradiation (1 pulse) of the [P<sub>0/3</sub> − 3H]<sup>3−</sup> at *m/z* 850.4 with a laser tuned at (A) 266 nm with an energy-per-pulse of 1.4 mJ and (B) 365 nm with an energy-per-pulse of 0.75 mJ.</figcaption></figure>
was due to higher energy photons delivered by the 266 nm laser that led to populated photo-excited states with different charge distribution and overall electronic nature. With 365 nm laser pulses, photo-cleavage of one o-nitrobenzyl group starts to occur with a single pulse, yet to a very moderate extent ($P_{1/3}^{3-}$ at m/z 805.4, ~6% of the precursor ion; Figure 2B). Very weak double photo-cleavage occurs ($P_{2/3}^{3-}$ at m/z 760.4, ~1% of the precursor ion; Figure 2B), but these data do not evidence the triple photo-cleavage to yield the targeted $P_{3/3}^{3-}$ ion expected at m/z 715.4. Experimental limitation at 365 nm did not allow us to explore higher energies per pulse. However, because no undesired competitive secondary fragmentation was observed at this wavelength, the enhancement of the yield of side-chain cleavage could be safely explored by multiple pulse accumulation (Figure 3). The number of pulses had to be multiplied by 10 to initiate significant levels of the first o-nitrobenzaldehyde loss ($P_{1/3}^{3-}$ at m/z 805.4, ~40% of the precursor ion, Figure 3B). It required a further increase to 100 for efficient production of the targeted $P_{3/3}^{3-}$ detected at m/z 715.3, together with significant precursor depletion (Figure 3C). Notably, no 135 Da neutral loss is observed upon irradiation of the $P_{0/0}^{3-}$ species in the same conditions (Figure S4C, supporting information), hence confirming that p-nitrobenzyl moieties are photo-inert as previously observed when UV irradiation was performed in solution [22].

Detailed inspection of the data indicates two additional features. On the one hand, occurrence of photo-detachment is revealed by signals of weak intensity (~1% major ion) in the 1100–1300 m/z range (which was not noticed at 266 nm). These $[P_{1/3} - 3H]^2-$ ions are detected for $P_{0/3}$ at m/z 1275.6, $P_{1/3}$ at m/z 1208.0 and $P_{2/3}$ at m/z 1140.5 (Figure S5, supporting information). As previously demonstrated for other types of polymers [35–37], the generation of radical ions could have been an interesting option to investigate alternative sequencing of digital oligomers in so-called activated electron photodetachment dissociation (a-EPD) mode. Yet, these radical anions are of too

low abundance for further useful CID experiments, preventing us to explore their dissociation behavior. Moreover, no radical anion was detected for the fully edited $P_{3/3}$ species. On the other hand, all $P_{1/3}$ anionic species are observed to eliminate water (Figures 2B and 3C). These water losses are already observed with single pulse irradiations (abundance of ~1% of the precursor ion), which suggests that this second photo-fragmentation pathway is in competition with the release of o-nitrobenzaldehyde. Ratios measured in the single-pulse photo-fragmentation mass spectrum (Figure 2B) suggest that the o-nitrobenzyl group loss is significantly favored (85%) with regard to dehydration (15%). However, the dehydration peaks reach significant intensities (25%–30% of fragment peaks) as many pulses are accumulated (Figure 3C). This could indicate that dehydration occurs from the ground state after internal conversion and vibrational heating, a process that is favored as the internal energy of the ions increases along with the multiple absorptions required to successively deplete the oligomer from its o-nitrobenzyl side-groups. Similar sequential heating has been reported previously in the gas phase [38]. No dehydration product was ever observed when these oligomers were revealed in solution [22], consistent with the fact that such internal energy accumulation is much less favored in a condensed medium, which favors fast energy dissipation. Interestingly, the $P_{0/3}^{3-}$ precursor anion displays multiple dehydration peaks (Figure 3C), including traces of $[(P_{0/3} - 3H_2O) - 3H]^3-$ at m/z 832.3, in which the number of dehydration steps equals the number of o-nitrobenzyl side-groups. On the opposite, data recorded in the same conditions for the $P_{0/0}^{3-}$ precursor anion only show traces of a unique water loss (Figure S4C, supporting information). Moreover, the fully revealed $P_{3/3}^{3-}$ ion barely displays traces of a single dehydration step, while $P_{2/3}^{3-}$ and $P_{1/3}^{3-}$ ions respectively undergo 1 and 2 water losses (Figure 3C). In other words, the propensity of $P_{1/3}^{3-}$ species to eliminate water molecules appears to be

<figure><img src="image_7.png" /><figcaption>**FIGURE 3** | LID spectra recorded upon irradiation of $[P_{0/3} - 3H]^3-$ (m/z 850.4, blue star) after (A) 1 pulse, (B) 10 pulses, or (C) 100 pulses with the laser tuned at 365 nm and 0.75 mJ per pulse.</figcaption></figure>
closely related to the number of *o*-nitrobenzyl units still present in the structure.

Lastly, it is possible to generate significant amounts of $P_{3/3}^{3-}$ (Figure 3C) and still without formation of low m/z irrelevant fragments (Figure S5, supporting information), but only when irradiating ions with a very large number of laser pulses (here 100 pulses). With our laser at 10 Hz (i.e., 100 ms inter-pulses period), irradiating ions with 100 pulses requires trapping of precursor ions for 10 s. This irradiation time is very long compared with MS timescales, which represents a major drawback that hampers the perspectives of applications beyond the proof of principle under these experimental conditions. Nevertheless, these preliminary experiments show that optimal photolysis of *o*-nitrobenzyl moieties can be performed at rather low power irradiation when compensated with long irradiation times. This suggests that LID could be more conveniently performed using a continuous wave (CW) UV laser.

## 3.3 | LID With CW Laser

Experiments were then performed using a CW laser emitting at 375 nm. This wavelength is also close to irradiation conditions performed in solution and should not lead to undesired low mass fragments. Figure 4 shows LID data recorded for $[P_{0/3} - 3H]^{3-}$: the three *o*-nitrobenzyl groups can be cleaved after 200 ms irradiation (Figure 4A) but more efficient photolysis of all *o*-nitrobenzyl groups is achieved when increasing the irradiation time to 1500 ms, as shown in Figure 4B with major production of $[P_{3/3} - 3H]^{3-}$ at m/z 715.3 and complete disappearance of its precursor. The full cleavage yield is comparable between Figure 4A (200 ms irradiation) and Figure 3C (10 s irradiation with pulsed laser at 365 nm), which indicates that under our experimental conditions (0.75 mJ pulses and 100 mW CW laser), the CW setup is ~50 times more efficient to edit the binary sequence of these oligomers.

<figure><img src="image_8.png" /><figcaption>**FIGURE 4** | LID spectra recorded upon irradiation with the CW laser (375 nm, 100 mW) of $[P_{0/3} - 3H]^{3-}$ (A and B) and $[P_{0/6} - 3H]^{3-}$ (C and D), both precursor ions at m/z 850.4. Irradiation time was successively set at 200 ms (A and C) and 1500 ms (B and D). The groups of peaks systematically correspond to a $P_{1/3}^{3-}$ fragment (with $i \le j$) together with one or multiple water losses. Red dotted lines are meant to show that, from incompletely revealed oligomer ions, only fully dehydrated products remain after 1500 ms irradiation.</figcaption></figure>
Successful cleavage is also demonstrated when increasing the number of o-nitrobenzyl groups in the precursor ion, as exemplified with efficient photo-edition of P<sub>0/6</sub><sup>3-</sup> into P<sub>6/6</sub><sup>3-</sup> at *m/z* 580.1 (Figure 4C,D). An irradiation time as short as 200 ms is here again efficient enough to generate the targeted P<sub>6/6</sub><sup>3-</sup> species as one of the major ions, whereas a single 365 nm pulse at 0.75 mJ hardly allowed the P<sub>1/6</sub><sup>3-</sup> to be formed (Figure S3H, supporting information). Moreover, even at the longest irradiation time and consistent with *p*-nitrobenzyl groups being photo-inert, the number of 135 Da losses from P<sub>0/j</sub><sup>3-</sup> is never greater than *j* (i.e., 3 from P<sub>0/3</sub><sup>3-</sup> in Figure 4B and 6 from P<sub>0/6</sub><sup>3-</sup> in Figure 4D). Thus, CW laser irradiation was observed to outperform pulsed laser irradiation by multiple orders of magnitude in terms of photo-cleavage yield, regardless of the number of *o*-nitrobenzyl groups to be cleaved in the oligomer. Of note, the amount of photodetachment remains extremely low under CW irradiation, regardless of irradiation times.

On the dehydration front, observations confirm the relationships previously established between occurrence of water loss and presence of *o*-nitrobenzyl groups. In particular, it is again observed that the fully edited P<sub>j/3</sub><sup>3-</sup> species are never dehydrated contrary to partially revealed products that still possess intact light-sensitive units (Figure 4). Moreover, after 1500 ms irradiation time (Figure 4B,D), a saturation regime is reached where mostly the fully revealed oligomers remain, together with a few dehydration products formed after P<sub>1/j</sub><sup>3-</sup> ions have eliminated (*j* − *i*) water molecules. This suggests that each dehydration event would prevent the loss of one *o*-nitrobenzaldehyde: this could either be due to the loss of absorption cross-section associated with the potential modification of optical properties of the dehydrated side-groups or to the structural modification hindering its release. Although not fully understood at this stage, these photo-induced dehydration reactions remain minor events, even for oligomers containing a large number of *o*-nitrobenzyl side-groups.

To tentatively identify optimal experimental conditions, evolution of the measured population of unrevealed towards fully revealed oligomer ions was plotted as the function of the CW irradiation time (Figure 5).

Within less than 300 ms, P<sub>3/3</sub><sup>3-</sup> becomes the most abundant ion and a plateau is reached within 400 ms (in green, Figure 5A), which means that shorter irradiation times may be sufficient to identify the fully edited species and generate enough of it for further isolation and CID sequencing. The P<sub>0/6</sub><sup>3-</sup> precursor requires 600 ms irradiation before the targeted P<sub>6/6</sub><sup>3-</sup> becomes the major ion on the LID spectrum (in pale blue, Figure 5B), and a plateau is reached around 900 ms. As a rough approximation from these experiments, ~100 ms irradiation per ortho units is requested to yield the desired photo-product as the major ion and ~130 ms irradiation per ortho units to reach the plateau. Extrapolating these values indicates it would require 800 ms to edit a full ortho P<sub>0/8</sub><sup>3-</sup> oligomer (and ~1050 ms to reach the plateau). Most importantly, it was seen from Figure 4A,C that 200 ms irradiation of P<sub>i/j</sub><sup>3-</sup> is enough to safely identify the fully edited P<sub>j/j</sub><sup>3-</sup> oligomer and to determine the original number *j* of *o*-nitrobenzyl side-groups from the number of ~135 Da shifts between the precursor ion and the final products. Accordingly, these conditions have been used when implanting CID to decode the LID-edited binary sequence.

## 3.4 | MS/MS Sequencing of LID-Edited Polymers

The last step of the analytical workflow consists of decoding the 0/1 binary sequence edited by LID in the gas-phase oligomer. To do so, a CID-MS³ stage was performed directly after the LID-MS² step. This is exemplified in Figure 6 with the P<sub>0/6</sub><sup>3-</sup> primary precursor ion (*m/z* 850.4) irradiated for 200 ms in the MS² stage to yield the photo-edited P<sub>6/6</sub><sup>3-</sup> ion at *m/z* 580.4, which was selected

<figure><img src="image_9.png" /><figcaption>**FIGURE 5** | Breakdown curves of (A) P<sub>0/3</sub><sup>3-</sup> and (B) P<sub>0/6</sub><sup>3-</sup> precursor ions as a function of CW irradiation time in LID.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**FIGURE 6** | $MS^3$ of the $P_{0/6}^{3-}$ ion at *m/z* 850.4, with the LID-edited $P_{6/6}^{3-}$ at *m/z* 580.4 (designated by a blue star) further submitted to CID for sequencing. Internal fragments are shown in grey, including deprotonated 1-bit monomer at *m/z* 288.0.</figcaption></figure>

as the secondary precursor for the CID-MS³ stage. As previously reported [34, 39], dissociation of sequence-defined poly(phosphodiester) chains proceeds via efficient cleavage of all bonds of the backbone phosphate groups, yielding α-containing (i.e., $a_i^{z-}$, $b_i^{z-}$, $c_i^{z-}$, $d_i^{z-}$) and ω-containing (i.e., $w_i^{z-}$, $x_i^{z-}$, $y_i^{z-}$, $z_i^{z-}$) product ions (Scheme S1, supporting information), named after the nomenclature proposed by McLuckey et al. for fragments of small multideprotonated oligonucleotides [40]. As shown in Figure 6, nearly all fragments expected in the eight series are detected with significant abundance although the collisionally activated $P_{0/6}^{3-}$ ion has been generated upon irradiation of the $P_{0/6}^{3-}$ ion during 200 ms only (that is, far from the optimal yield defined in Figure 5B). Here, the 01000001 binary sequence of the LID-edited $P_{6/6}^{3-}$ is readily reconstructed from the left-hand side using α-containing fragments or from the right-hand side employing ω-ended product ions (as detailed in Table S1, supporting information). In summary, these data enable full sequence coverage with high confidence, similar to CID data that were recorded off-line with light-edition of oligomers in solution [22], yet in a much higher throughput workflow.

## 4 | Conclusions

The development of polymer encoding techniques attracts an increasing interest, notably when macromolecular media enable additional levels of confidentiality, which also rises the demand for efficient and rapid decoding methods. In this paper, the possibility to include both the editing and decoding steps requested to retrieve information stored in phosphodiester octamers in a single gas-phase mass spectrometry workflow was examined with an ion trap instrument equipped with various light sources. Nanosecond-pulsed laser and CW laser irradiations were compared. The pulsed laser failed at fully dissociating all photo-sensitive o-nitrobenzyl side-groups of mass-selected oligomers and generated high levels of small fragments irrelevant for the targeted revelation. On the contrary, use of

a CW laser at 375 nm enabled efficient photo-cleavage of all o-nitrobenzyl side-groups within MSⁿ compatible irradiation times. Polymer dehydration appeared as a minor competitive reaction compared with side-chain losses, which remained the dominant photo-fragmentation pathway. Overall, an irradiation time of 300 to 400 ms was enough to observe full dissociation of photo-sensitive side-groups in all examined octamers. However, saturation of the fully edited oligomer may be attained with as long as 1 to 1.5 s depending on the number of o-nitrobenzyl side-groups to cleave, but such a level of production was not mandatory to further engage the edited ion into a successful CID sequencing step. It was shown that a simple MS² stage with CW laser irradiation enabled direct determination of the number of photosensitive units that determines the number of 0-bits in the fully edited octamer. Subsequent selection and CID of the light-edited oligomer was performed and permitted to retrieve encoded binary information via MS/MS sequencing. These results constitute the first proof-of-principle that both editing and decoding are feasible in a single MS³ experiment in an ion trap, relieving the need for time-consuming solution phase edition prior to the MS/MS reading step.

### Associated Content

Fragment nomenclature; MS and MS/MS of octamers prior light-edition; LID spectra of $P_{0/3}^{3-}$ at 266 nm and of $P_{0/6}^{3-}$ at 365 nm; structure proposed for small fragments observed at 266 nm; LID spectra of $P_{0/0}^{3-}$ at 266 nm and at 365 nm; LID spectra of $P_{0/0}^{3-}$ with 100 pulses at 365 nm; sequencing fragments of $P_{6/6}^{3-}$.

### Acknowledgments

L.C. acknowledges support from Spectropole, the Analytical Facility of Aix-Marseille University, by allowing a special access to the instruments
purchased with European Funding (FEDER OBJ2142-3341). The authors thank Dr Marion Girod from the Institut des Sciences Analytiques of University Lyon 1 for the courtesy of the CW laser at 375 nm. Niklas Felix König and Abdelaziz Al Ouahabi are thanked for the synthesis of the oligomers.

## Conflicts of Interest

The authors declare no conflicts of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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## Supporting Information

Additional supporting information can be found online in the Supporting Information section.