Mass spectrometry sequencing of long digital polymers facilitated by programmed inter-byte fragmentation

Abstract

In the context of data storage miniaturization, it was recently shown that digital information can be stored in the monomer sequences of non-natural macromolecules. However, the sequencing of such digital polymers is currently limited to short chains. Here, we report that intact multi-byte digital polymers can be sequenced in a moderate resolution mass spectrometer and that full sequence coverage can be attained without requiring pre-analysis digestion or the help of sequence databases. In order to do so, the polymers are designed to undergo controlled fragmentations in collision-induced dissociation conditions. Each byte of the sequence is labeled by an identification tag and a weak alkoxyamine group is placed between 2 bytes. As a consequence of this design, the NO-C bonds break first upon collisional activation, thus leading to a pattern of mass tag-shifted intact bytes. Afterwards, each byte is individually sequenced in pseudo-MS³ conditions and the whole sequence is found.Digital information can be stored in monomer sequences of non-natural macromolecules, but only short chains can be read. Here the authors show long multi-byte digital polymers sequenced in a moderate resolution mass spectrometer. Full sequence coverage can be attained without pre-analysis digestion or the help from sequence databases.

Fig. 1

General concept studied herein for the sequencing of long digital polymer chains. a Molecular structure of the sequence-coded polymers prepared by automated phosphoramidite chemistry. These digital polymers contain n + 1 coded bytes noted in red. A byte is a sequence of eight coded monomers that represent 8 bits. Two consecutive bytes are separated by a linker noted in black, which contains a NO-C bond that can be preferentially cleaved during MS/MS analysis. In order to sort out the bytes after MS/MS cleavage, n bytes of the sequence are labeled with a mass tag noted in blue. b Molecular structure and mass of the two coded synthons that define the binary code in the polymers. c Molecular structure and mass of the mass tags that are used as bytes labels. In order to induce identifiable mass shifts after MS/MS cleavage, the mass of a byte tag (noted in blue) shall not be a multiple of 28, which is the mass difference between a 0 and a 1 coded unit. In addition, the mass difference between two tags (noted in grey) shall not be a multiple of 28. d Schematic representation of the mass spectrometry sequencing of a digital polymer containing 4 bytes of information. The polymer is first analyzed in MS/MS conditions, which lead to the favored cleavage of the weak NO-C bonds (depicted in yellow inside the grey spacers). Since they carry mass tags, the resulting cleaved bytes are sorted out by mass in the MS/MS spectrum (the displayed MS² cartoon is idealized for clarity). Afterwards, each byte can be easily sequenced in MS³ conditions and the whole binary sequence can be deciphered

Fig. 2

Sequencing of a 4-byte digital polymer that contains the ASCII-encoded word Byte. a High-resolution electrospray mass spectrum (MS¹) obtained in the negative ion mode for a 4-byte digital polymer (Supplementary Table 1, Entry 5). The upper numbers represent the different charge states observed for the polymer. Dark grey diamonds indicate in-source fragments (see Supplementary Table 2 for a detailed interpretation of each peak) and dark grey squares designate different charge states of two different synthesis impurities. b MS² spectrum (0.56 eV, center of mass frame) obtained by collision-induced dissociation of the [M-12H]¹²⁻ precursor ion, which carries on average three charges per byte. In this case, the MS/MS single-byte, double-byte, and triple-byte fragments are predominantly observed as trianions, hexa-anions, and nona-anions, respectively. Other charge states can be observed to a minor extent and are denoted by dark grey circles (see Supplementary Table 2 for a detailed interpretation of each peak). c Molecular sequencing (pseudo-MS³) of a byte fragment obtained by collision-induced dissociation (0.56 eV, center of mass frame) of the precursor trianion [M-3H]³⁻. For clarity, only the sequencing of byte 4 is shown as an example in this figure. The sequencing of bytes 1–3 is shown in Supplementary Fig. 6

Fig. 3

MS² spectrum of an 8-byte digital polymer containing the ASCII-encoded word Sequence. This spectrum (0.59 eV, center of mass frame) was obtained by collision-induced dissociation of the [M-24H]²⁴⁻ precursor ion of polymer 11 in Supplementary Table 1. In this case, the MS/MS single-byte, double-byte, triple-byte, and tetra-byte fragments are predominantly observed as trianions, hexa-anions, nona-anions, and dodeca-anions, respectively. Other charge states can be observed to a minor extent and are denoted by dark grey circles (see Supplementary Figs. 21b and 22 for a detailed interpretation of each peak). The subsequent pseudo-MS³ sequencing of all bytes is shown in Supplementary Figs. 23–25