Revealing data encrypted in sequence-controlled poly(alkoxyamine

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Revealing data encrypted in sequence-controlled poly(alkoxyamine phosphodiester)s by combining ion mobility with tandem mass spectrometry Jean-Arthur Amalian, Gianni Cavallo, Abdelaziz Al Ouahabi, Jean-Francois Lutz, and Laurence Charles Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00813 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 11, 2019

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Analytical Chemistry

Revealing data encrypted in sequence-controlled poly(alkoxyamine phosphodiester)s by combining ion mobility with tandem mass spectrometry Jean-Arthur Amalian,1 Gianni Cavallo,2 Abdelaziz Al Ouahabi,2 Jean-François Lutz,2* Laurence Charles1* 1Aix

Marseille Université, CNRS, UMR 7273, Institute of Radical Chemistry, 13397,

Marseille Cedex 20, France. 2Université

de Strasbourg, Institut Charles Sadron, UPR22-CNRS, BP84047, 23 rue du Loess,

67034 Strasbourg Cedex 2, France. ABSTRACT The defined sequence of two co-monomers in sequence-controlled macromolecules can be used to store binary information which is further decoded by MS/MS sequencing. In order to achieve the full sequence coverage requested for reliable decoding, the structure of these polymers can be optimized to minimize their dissociation extent, as shown for poly(alkoxyamine phosphodiester)s (PAPs) where weak alkoxyamine bonds were introduced in each repeating unit to make all phosphate groups MS/MS silent. However, for secret communications, a too high MS/MS readability could be a drawback. In this context, the design of PAPs was further optimized in this work to also include a decrypting key based on slight variation of fragment collision cross section. This was achieved by employing two different nitroxides to build the alkoxyamine moiety, each containing a coding alkyl segment of the same mass but different architectures. As a result, the digital sequence determined from primary fragments observed in MS/MS had to be decrypted according to appropriate rules that depend on the drift times measured by ion mobility spectrometry for repeating units released as secondary product ions.

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INTRODUCTION Digital polymers are synthetic macromolecules with a controlled sequence of two comonomers that can be defined as the 0- and 1-bit of the ASCII code to store binary information at the molecular level,1-3 as already done in the field of DNA storage with individual chains containing about 100 coded residues.4 As long as they are composed of co-monomers of different mass, such information can be deciphered by tandem mass spectrometry (MS/MS) sequencing, as popularized by the field of proteomics where this technique is routinely used to retrieve the nature and relative location of building units in peptides.5 In recent years, MS/MS sequencing was successfully applied to read information stored in various abiological polymers.6-14 However, in contrast to the case of peptides that can be identified by combining a partial sequence of amino acids with appropriate database searching,15 full sequence coverage is required for reliable reading of coded messages written in sequence defined synthetic polymers. This is indeed a mandatory issue when using the 8-bit ASCII alphabet, where each letter is coded by a set of 8 bits which only differs by the location of one digit compared to the following letter (e.g., "a": 01100001 versus "b": 01100010). To achieve full coverage of a copolymer sequence, the same dissociations should proceed with similar efficiency at all units. Alternatively, the original primary structure can be reconstructed by combining partial sequences built from fragments that contain one or the other chain end. However, in both cases, the number of dissociation reactions should be limited in order to avoid dilution of the whole signal over multiple series of fragments, which decreasing abundance as the chain size increases also raises major issues for automated decoding using appropriate software.16, 17 The dissociation behavior of polymeric chains is primarily dictated by the types of bonds composing their skeleton,18 although the nature of dissociating ions19 as well as the implemented activation method20-22 also influence MS/MS outputs. Digital polymers produced in our group span a wide range of fragmentation behaviors, as observed when using collision induced dissociation (CID). The most easy-to-read species were undoubtedly sequenced-defined polyurethanes:8 they experienced only one bond cleavage per unit and, because they were ionized by deprotonation of their α end-group,23 this led to a single series of product ions spaced by the mass of one or the other coded monomer. Accordingly, binary information coded in these species could literally be read from MS/MS data. In contrast, synthesis of sequenced-controlled poly(phosphodiester)s could be automated to produce very long monodisperse chains24 but their 2 ACS Paragon Plus Environment

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dissociation was observed to proceed via efficient cleavage of all phosphate bonds contained in each unit.11 As a result, their MS/MS spectra exhibited four sets of complementary product ions, offering rich but highly redundant sequence information spread over multiple signals. Moreover, spectral complexity further increased with the precursor ion size because poly(phosphodiester)s were readily produced as highly charged species in the negative ion mode electrospray ionization (ESI) and hence generate fragments also dispersed in terms of charge state. In this context, the upper limit for full sequence coverage was estimated to be a degree of polymerization (DP) of about 50.25 However, the structure of sequence-controlled polymers can be optimized to simplify their dissociation behavior and hence improve their MS/MS readability.26 For example, poly(alkoxyamine phosphodiester)s (PAPs) were conceived from poly(phosphodiester)s by placing an alkoxyamine moiety between all phosphodiester coding units.9 As expected from this structural design, the low-energy homolysis of C–ON bonds made all phosphate bonds MS/MS silent and occurred in each repeating unit, yielding a single set of complementary fragments from which the co-monomeric sequence of any PAPs could be readily reconstructed.25, 26 Using the nitroxide as an additional coded moiety further permitted to increase the storage capacity of PAPs, with two bits of information in each repeating unit, while keeping the same simple MS/MS pattern.17 While the simplicity of CID pattern is a clear advantage to achieve full sequence coverage of digital polymers, it could be a real drawback when such macromolecules are to be used for secret communications. Indeed, when sequencing rules are too obvious, the binary sequence of digital polymers can be deciphered using commercial instrumentation available nowadays in most laboratories. To provide an additional security level to information-containing polymers, we conceived an encrypting key based on slight conformational variation of the coding nitroxide in PAPs. As a result, retrieving information written in the backbone of these polymers can no longer be done by MS/MS alone but requires a more sophisticated coupling involving ion mobility spectrometry (IMS) to identify the key for subsequent decryption of the coded message revealed by MS/MS sequencing. EXPERIMENTAL SECTION Chemicals. Sequence-controlled PAPs were synthesized according to a previously reported protocol,17 via an orthogonal iterative pathway which implies successive phosphoramidite and 3 ACS Paragon Plus Environment


radical-radical coupling steps. The selected set of PAPs used in this study is listed in Table S1 (Supporting Information). It shall be noted that the polymers prepared with the alphabet 0’b/1’b are obtained in moderate yields and contain a significant amount of truncated sequences, as described in our previous publication.17 Still, the targeted digital sequence can be detected as the major species in these samples and therefore be easily decrypted by MS/MS-IMS. PAPs were solubilized in methanol (SDS, Peypin, France) and further diluted in a methanolic solution of ammonium acetate (3 mM; Sigma-Aldrich, Saint Louis, MO). Sodium acetate and poly(methylmethacrylate) (PMMA) used for external TOF calibration in the MS mode were from Sigma Aldrich. Mass spectrometry and ion mobility spectrometry. High resolution MS, MS/MS and traveling wave ion mobility spectrometry (TWIMS) experiments were performed with a Synapt G2 HDMS instrument from Waters (Manchester, UK). The electrospray ionization (ESI) source was operated in the negative ion mode with the following settings: capillary voltage, –2.27 kV; sampling cone voltage, –20 V; desolvation gas (N2) flow, 100 L h-1 at 35°C; source temperature, 35°C. A syringe pump was used to introduce PAP sample solutions in the ESI source at a 10 μL.min-1 flow rate. This instrument includes a quadrupole (used for ion selection prior activation), a mobility cell and an orthogonal acceleration time-of-flight (oa-TOF) mass analyzer. Ions were all accurately mass measured using the oa-TOF, either in the MS mode when arising from the ESI source or in the MS/MS mode when generated as fragments upon collision-induced dissociation (CID, argon) of quadrupole-selected precursor ions in the ion trap device located in front of the mobility cell. In MS/MS-IMS experiments, product ions were subjected to an ion mobility step prior being injected in the oa-TOF for mass analysis. To do so, they were ejected from the ion trap into a cooling cell (helium flow: 180 mL.min-1) prior reaching the TWIMS cell filled with N2 (3.45 mbar) and operated in various wave velocity (m.s-1)/wave height (V) conditions (see text). In the MS mode, external calibration of the oa-TOF was performed using oligomers of PMMA adducted with sodium, while the precursor ion was used as an internal standard in the MS/MS mode. Instrument control, data acquisition and data processing of all experiments were achieved with the MassLynx 4.1 programs provided by Waters. Charge state deconvolution of MS/MS data was performed with MagTran (Amgen Inc.), an implementation of the ZSCORE algorithm.27


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RESULTS & DISCUSSION Coding systems in PAPs. The general structure of PAPs is depicted in Scheme 1a. Each repeating unit is composed of a phosphodiester moiety connected to a nitroxide, both containing a small alkyl segment (Ra and Rb, respectively) to implement a binary code based on mass. As detailed in Scheme 1b, coded phosphodiesters were named 0a (223.1 Da) or 1a (251.1 Da) depending on Ra being a propyl or a 2,2-dimethyl propyl segment, respectively. Two sets of coded nitroxides have been used, with Rb segments of different length. The first set involves a propyl group which holds either one (for 0) or two (for 1) methyl substituants on the first carbon (Scheme 1c, left): these nitroxides were named 0'b and 1'b, respectively. The second set is based on a butyl segment with either no (for 0) or one (for 1) methyl group on the second carbon (Scheme 1c, right): these nitroxides were named 0"b and 1"b, respectively. Accordingly, mass of 0b is 254.2 Da and that of 1b is 268.2 Da, regardless of the architecture of their alkyl segment. It should be emphasized here that while all PAPs have the same coding system for Ra, they include either one or the other nitroxide set. In other words, the two alphabets available for the Rb part were never mixed inside a given chain.

Scheme 1. a) Schematic structure of sequence-controlled PAPs and cartoon showing the distribution of Ra- (in red) and Rb- (blue) containing bits relative to cleavable C–ON bonds (in yellow). Molecular structure and mass of b) phosphodiester segments (0a/1a) and c) nitroxide moieties constructed with either the propyl (0'b/ 1'b) or the butyl (0"b/ 1"b) skeleton.



MS/MS sequencing of PAPs. Collisional activation of deprotonated PAPs induced competitive homolytic cleavages of all alkoxyamine bonds, yielding two product ion series named c when containing the α end-group or y when still holding the  end-group.9 When selecting precursor ions with all phosphate groups deprotonated, C–ON bond homolysis generate ci•i– and yi•i– fragments which charge state i is equal to the number of phosphodiester groups they contain (Figure 1a).26 These conditions prevent charge state dispersity of product ions, which highly simplifies MS/MS data. This is illustrated in Figure 1b with the PAP polymer P4 coding for 10001000; as it was constructed with the 0'b/1'b nitroxide set, the sequence should be chemically described as 1a0'b0a0'b1a0'b0a0'b. These CID data show that the fully deprotonated precursor ion at the –4 charge state (m/z 509.3) dissociates to form ci•i– ions, observed on the left-hand side of the spectrum and having their m/z values increasing with i, and yi•i– observed on the right-hand side with m/z values decreasing as i increases.

Figure 1. a) Structure and fragmentation scheme of the PAP polymer P4 (2041.2 Da) of sequence 1a0'b0a0'b1a0'b0a0'b. b) MS/MS of the quadruply deprotonated P4 PAP (m/z 509.3) using a 0.70 eV collision energy (center-of-mass frame). Stars designate internal fragments detailed in the enlarged 476-506 m/z range shown in inset. c) Spectrum obtained after charge state deconvolution of the ci•i– ion series and used to reconstruct the binary sequence.


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Full coverage of the sequence could be achieved when analyzing the sole ci•i– ion series, either "manually" according to the calculation procedure described in Supporting Information or in an automated manner with the MS-DECODER software.17 Alternatively, charge deconvolution of these fragments allowed convenient visualization of the sequence, as illustrated Figure 1c: the first bit is identified from the c1 peak measured at the mass of αXa (α0a: 282.1 Da, α1a: 310.1 Da), the distance measured between any two consecutive peaks is equal to the mass of one X'bXa dyad (0'b0a: 477.3 Da, 1'b0a: 491.3 Da, 0'b1a: 505.3 Da, 1'b1a: 519.3 Da), and the last bit is determined from the mass difference between the intact PAP chain and the last c since it corresponds to the mass of X'b (0'b: 271.2 Da, 1'b: 285.2 Da). Owing to the dissociation mode of deprotonated PAPs and because MS/MS sequencing is (by principle) based on mass, the same MS/MS data as shown in Figures 1b-c were obtained for the 1a0"b0a0"b1a0"b0a0"b (Figure S1, Supporting Information). In other words, without prior knowledge of the synthesis history, MS/MS cannot distinguish 1a0'b0a0'b1a0'b0a0'b from 1a0"b0a0"b1a0"b0a0"b. While the two coding systems (0'b/1'b vs 0"b/1"b) used for nitroxide moieties cannot be identified based on mass, they exhibit structural differences (Scheme 1c) that might influence their collision cross section (CCS). This parameter influences the ion drift time (tD) as measured in IMS, a separation technique based on differential mobility of ions as they travel through a buffer gas under the influence of an electric field.28,

29

For example, IMS was reported to

differentiate synthetic polymers with isomeric pending groups such as poly(n-butylmethacrylate) and poly(tert-butylmethacrylate).30 ESI-MS/MS-IMS experiments were hence performed to try to distinguish fragments generated from PAP isomers prepared with a different Xb alphabet. Ion mobility spectrometry of PAP fragments. The instrumental configuration of the mass spectrometer used here offers the possibility to perform IMS experiments of fragments as the mobility cell is placed after an ion trap device, where ionic species generated in the ESI source and subsequently selected in a quadrupole mass analyzer can be collisionally activated. When performing such MS/MS-IMS experiments, ci•i– or yi•i– ions generated from PAPs containing X'b were found to exhibit the same mobility as those formed from PAPs built with X"b segments, regardless of the experimental conditions operated for the TWIMS cell (data not shown). As the sole architectural difference between 0'b and 0"b or between 1'b and 1"b isomers is a branched CH3 moiety (Scheme 1c), these results would indicate a very low contribution of this methyl substituent to the overall conformation of the quite large ci•i– or yi•i– ions (the smallest fragment 7 ACS Paragon Plus Environment


containing one Xb segment is c2•2–, which mass ranges from 759.4 Da for α0a0b0a to 829.4 Da for α1a1b1a). However, beside ci•i– or yi•i– ions, CID of deprotonated PAPs always generated additional product ions of much lower size than the c2•2– fragment. These ions, designated with stars in Figure 1b, are internal fragments that, by definition, are useless for sequencing purposes since they do no longer contain any of the original chain ends. These are secondary ions formed upon C–ON bond homolysis in ci•i– (except c1•1–) or yi•i– and can hence be described as deprotonated biradicals composed of XbXa dyads, the number of which is identified by the ion charge state. As shown in the inset of Figure 1b, the signal at m/z 476.3 corresponded to the deprotonated 0'b0a dyad while the peak at m/z 504.3 was the deprotonated 0'b1a dyad. Based on their respective isotopic pattern, the m/z 490.3 ion was identified as the doubly charged 0'b0a0'b1a species and the m/z 485.6 ion was assigned to the triply charged 0'b0a0'b1a0'b0a biradical. Since the limited size of deprotonated XbXa dyads might allow the conformational contribution of one branched methyl group to become significant, IMS was then evaluated for its performance at discriminating X'bXa from X"bXa. In order to measure the four possible XbXa combinations for internal fragments, PAPs with different sequences were prepared with either 0'b/1'b or 0"b/1"b nitroxides (Table S1, Supporting Information). A total of 21 different sets of TWIMS experimental conditions (wave velocity, VW/wave height, WH) were then tested in the ESI-MS/MS-IMS instrumental configuration to measure drift times (tD) of internal fragments (Tables S2 and S3, Supporting Information). In these experiments, activation energy of deprotonated PAPs was raised (compared to MS/MS conditions used for sequencing purposes) in order to promote secondary dissociations and hence maximize the abundance of internal fragments. Since the uncertainty associated with tD measurements in our instrument is typically of ±0.07 ms, the minimal value for the drift time difference (ΔtD) between isomeric dyads to be significant should be higher than 0.14 ms. As shown in Figure 2a for the deprotonated 0b0a internal fragment (m/z 476.3), clear distinction was achieved between the two alphabets used to prepare the 0b segment, as measured by ΔtD = tD(0'b0a) – tD(0"b0a). More precisely, 90% of the tested WV/WH experimental conditions allowed ΔtD to be significant whereas it was below the 0.14 ms threshold (designated by the black dotted line in Figure 2a) in only two cases. Interestingly, all calculated ΔtD values were positive, indicating a slightly larger conformation of 0'b0a compared to 0"b0a. IMS traces obtained for


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these two internal fragments are compared in Figure 2b, using the most favorable experimental conditions (WV = 380 m.s-1, WH = 25 V) that allowed a maximal ΔtD of 0.32 ms for this dyad.

Figure 2. a) Drift time difference (ΔtD, ms) measured between 0'b0a and 0"b0a internal fragments (both detected at m/z 476.3) as a function of the wave velocity/wave height operating conditions of the TWIMS cell. Data are expressed as mean ± σ (6 replicates). The dotted line at ΔtD = 0.14 ms designates the threshold for ΔtD to be significant. b) ESI-MS/MS-IMS traces extracted at m/z 476.3 from PAP precursors containing the 0"b0a (top) or the 0'b0a motif (bottom), using a wave velocity of 380 m.s-1 and a wave height of 25 V.

Results of similar quality were obtained for internal ions of different composition and experimental conditions such as WV = 550 m.s-1 and WH = 30 V were found as a good compromise for all XbXa internal fragments (Table S4, Supporting Information). Using these TWIMS experimental settings, tD values typically expected for each dyad are listed in Table 1. Overall, these data indicate that performance of TWIMS is sufficient for the coding system used for the Xb segment to be determined from tD measured for internal fragments of PAPs, regardless of the sequence of the dissociating precursor ion. Therefore, one can envisage specific encryption rules to be associated with one or the other chemical alphabet used to prepare Xb nitroxide (i.e., the secret key), so that messages chemically encoded in the backbone of PAPs can only be read when combining IMS data (to identify the key with reference to Table 1) with MS/MS sequencing.



internal ion m/z 476.3 m/z 490.3 m/z 504.3 m/z 518.3

dyad 0'b0a 0"b0a 1'b0a 1"b0a 0'b1a 0"b1a 1'b1a 1"b1a

tD (ms) 7.06 6.78 7.27 6.97 7.60 7.32 7.70 7.49

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ΔtD (ms) 0.28 0.30 0.28 0.21

Table 1. Average drift times (tD, ms) measured in TWIMS (WV=550 m.s-1, WH=30 V) for all types of singly charged internal fragments generated from PAP polymers constructed with either 0'b/1'b or 0"b/1"b nitroxides. Uncertainty associated to tD values is ±0.07 ms.

MS/MS-IMS to decipher data encrypted in PAPs. In order for a receiver (or decryptor) to be able to read original messages written by the sender (or encryptor), both have to meet for preliminary exchanges (Figure 3): the decryptor receives i) the decoding rules (how the 0/1 binary code has been defined with regard to the mass of each repeat unit), ii) a set of polymer standards to optimize IMS experimental conditions to build his/her own reference data to identify the key as in Table 1 (eventually based on experimental conditions provided by the writer if both possess the same instrument) and iii) the decrypting rules (that could be modified at any time upon any further agreement between the writer and the reader).

Figure 3. Schematized workflow for efficient secret communications using PAPs. During preliminary exchanges, the writer (or encryptor) and the authorized reader (or decryptor) meet to agree on the decrypting process; the writer indicates the coding rules (how the 0/1 binary code is defined by repeating unit mass), provides a set of polymer standards and the MS/MS-IMS analytical set-up so that the reader can establish his/her own drift time reference data to identify the key hidden in the nitroxide moiety, and reveals the decrypting rules. 10 ACS Paragon Plus Environment

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Then, sequence-defined PAPs containing secret information are sent by the encryptor to the decryptor who applies the following two-step analytical methodology to decrypt the original message. First, soft CID (0.7-1.0 eV, center-of-mass frame) is performed for the fully deprotonated PAP precursor ion; amongst these MS/MS data, ci•i– ions are used to characterize a so-called primary sequence. Then, the same MS/MS experiment is repeated but i) using increased activation energy (2.5-3.0 eV, center-of-mass frame) to promote internal fragments and ii) operating the TWIMS cell. In this MS/MS-IMS experiment, drift times are measured for all internal fragments and compared to reference data (Table 1 or equivalent table established by the reader) to determine which alphabet was employed for the Xb-bits. Then, knowing the encryption rules from the writer, the reader can decrypt the primary code and obtain the original message. As an example, let’s consider the following simple decrypting rules: -

if the 0'b/1'b alphabet was used, then digits within each XbXa dyad should be inversed: such a dyad inversion would transform 0b1a into 10 and 1b0a into 01,

-

if the 0"b/1"b alphabet was used, then any 0"b becomes a 1-bit and any 1"b becomes a 0bit (so-called Xb bit inversion),

-

regardless of the alphabet used for Xb bits, Xa bits remain as determined by MS/MS (0a = 0 and 1a = 1).

Then, let’s apply these rules to decipher the digital message from spectral data shown Figure 4. Low energy (0.80 eV, center-of-mass frame) CID data recorded for [PAP – 4H]4– at m/z 505.8 allowed clear detection of all ci•i– and yi•i– product ions (Figure 4a), and charge deconvolution of ci•i– ions allowed the 0a1b0a0b1a0b0a0b primary sequence to be determined (Figure 4b). Raising the center-of-mass energy to 2.70 eV promoted consecutive dissociation of ci•i– and yi•i– ions and favored formation of internal fragments (Figure 4c). This CID spectrum shows that only one member of the primary ion series (i.e., c1•1–) survived these activation conditions, while the abundance of the expected singly charged internal fragments at m/z 476.3 (0b0a), m/z 490.3 (1b0a) and m/z 504.3 (0b1a) was significantly increased. Of note, the fourth ion designated with a star at m/z 497.3 in Figure 4a and no longer observed in Figure 4c was the doubly charged 1b0a0b1a internal fragment that has also undergone dissociation when collision energy was raised. Additional peaks detected in the 300-475 m/z range of Figure 4c revealed that cleavage of phosphate bonds became competitive in this energy regime. Since this higher energy MS/MS experiment was coupled with IMS, drift times could be measured for the three internal fragments: 11 ACS Paragon Plus Environment


comparing these results (Figure 4d) with data listed in Table 1 permitted to conclude that the 0"b/1"b alphabet was used to prepare this PAP. Since the key has been identified, appropriate decrypting rules (0"b → 1 and 1"b → 0) could be applied to translate the 0a1b0a0b1a0b0a0b primary sequence read in MS/MS into the 00011101 message.

Figure 4. a) MS/MS recorded at a 0.80 eV collision energy for the quadruply deprotonated PAP at m/z 505.8. b) Charge deconvolution of the ci•i– ion series, revealing the 0a1b0a0b1a0b0a0b primary sequence. c) MS/MS recorded at a 2.70 eV collision energy for [PAP – 4H]4– to promote formation of the three singly charged internal fragments (designated by stars). d) ESI-MS/MSIMS traces extracted at m/z 504.3 (top), m/z 490.3 (middle) and m/z 476.3 (bottom), using a wave velocity of 550 m.s-1 and a wave height of 30 V. Vertical dotted grey lines in part d) indicates expected tD for each deprotonated dyad. Collision energies expressed in the center-of-mass frame. In summary, as long as preliminary exchanges between the writer and authorized readers are kept secret (Figure 3), sequence-controlled PAPs allow different levels of security for any further confidential communications. Indeed, although it may be quite easy for an unauthorized reader to figure out that binary messages are exchanged based on MS/MS revealing two different repeating units, any adversary suspecting secret communication is happening would also have to know i) that a key is hidden within the sequence-defined polymer, ii) how to analyze the polymer to evidence the key and iii) which decrypting rules are to be associated with a given key.


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CONCLUSION Overall, these results clearly evidenced the potential of sequence-controlled PAPs for secure communications. An additional security level was implemented in these information-containing macromolecules by hiding a decrypting key in the coding nitroxide part of each repeating unit. This permitted to keep advantage of the high MS/MS readability of these sequence-controlled polymers while preventing any unauthorized readers to access messages encoded in their backbone. Indeed, both the decrypting rules and the appropriate IMS experimental conditions to identify the key have to be known for the original information to be accurately revealed. Interestingly, the key was identified here by IMS measurements performed for internal fragments that are useless for sequencing and hence usually never considered. More sophisticated encrypting rules can of course be envisaged based on coding theory and cryptography. In addition to encrypting purposes, these rules can also serve to artificially increase data storage capacity of small PAP chains, permitting to conceive messages composed of a number of bits which is larger than the number of coding segments. ASSOCIATED CONTENT Supporting Information. This Supporting Information is available free of charge on the ACS Publications website. Table S1, binary sequence of PAPs used in this study; Calculation procedure for MS/MS sequencing; Figure S1, MS/MS sequencing of P8; Table S2, drift times measured for X'bXa internal fragments; Table S3, drift times measured for X"bXa internal fragments; Table S4, average drift time differences, ΔtD = tD(X'bXa) – tD(X"bXa). AUTHOR INFORMATION Corresponding Authors. *E-mails: [email protected]; [email protected]. Author Contributions. L.C. designed the analytical study and wrote the manuscript. J.-F.L. conceived the coded polymers. J.-A.A. performed MS/MS and IMS experiments. G.C. and A.O.A. synthesized the polymers. All authors have given approval to the final version of the manuscript.



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). J.F.L. thanks the H2020 program of the European Union (project Euro-Sequences, H2020-MSCA-ITN-2014, grant agreement n°642083) and the French National Research Agency (ANR project 00111001, grant number ANR-16-CE29-0004-01) for financial support. The PhD position of G.C. is supported by the ITN Euro-Sequences and the post-doc position of A.A.O is supported by the ANR. REFERENCES (1)

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TOC graphic.



TOC graphic

ACS Paragon Plus Environment

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Scheme 1 254x190mm (96 x 96 DPI)



Figure 1


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Figure 2



Figure 3 177x73mm (96 x 96 DPI)


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Figure 4


Revealing data encrypted in sequence-controlled poly(alkoxyamine

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