MALDI-MS2 Analysis of Saponins

Saponin ion peaks were further analyzed using MS2 fingerprints generated with the collision-induced dissociation (CID) from their respective glycan structures. The techniques used are also able to distinguish the structural differences among the isomers following HPCPC separation. As a typical example, the MALDI-MS<sup>2</sup> fingerprints for the ion detected at *m/z* 1477.7 (triterpene glycoside) are shown in Figure 2. The schematic fragmentation of Lessonioside A as a representative is shown in Supplementary Figure S2. The fragmentation pattern of the sodiated compound at *m/z* 1477.7 in consecutive MS experiments is discussed in detail below for stepwise elucidation of the molecular structure of these compounds.

**Figure 2.** Positive tandem MALDI (matrix-assisted laser desorption/ionization) spectrum analyses of the precursor ion (saponin) detected at *m/z* 1477.7. The MS2 fragmentation profile of the ion at *m/z* 1477.7. Figure shows the collision-induced fragmentation of parent ions at *m/z* 1477.7. The full and dotted arrows show the possible fragmentation pathways of this ion using CID (collision-induced dissociation). The blue arrows show the fragmentation of the isomeric congeners Lessonioside A where the red arrows indicate the decomposition patterns of Lessonioside C. These analyses revealed that this ion corresponds to isomeric compounds.

CID activates three feasible independent fragmentation pathways of cationized parent ions shown in full and dotted arrows. First, as described in Figure 2, the consecutive losses of the deacetylated aglycone, acetic acid (AcOH), 3-*O*-methyl-D-glucose (MeGlc), D-quinovose (Qui), D-xylose (Xyl), MeGlc and Xyl residues (blue arrows) followed by D-glucose (Glc) yielded ion fragments at *m/z*

1007.5, 947.5, 771.4, 625.2, 493.1, 317.1 and 185.0 (Figure 3a), respectively, in one of the new isomers for which we propose the name Lessonioside A. The loss of aglycone (Agl) generated the ion at *m/z* 947, corresponding to the complete sugar moiety. The ion at *m/z* 493.1 corresponds to the diagnostic sugar reside [MeGlc-Glc-Xyl + Na]+. Further, the sequential losses of Glc and Xyl units from this key diagnostic peak (*m/z* 493.1) generated ions at *m/z* 331.1 and 199.0 (Figure 3b).

With another isomer, the consecutive losses of the deacetylated aglycone, MeGlc, Glc, Xyl and AcOH followed by the hydrated three sugar units (red arrows) produced ions at *m/z* 981.3, 805.3, 643.2, 511.2 and 451.1, respectively, (Figure 3c) revealed the structure of a second new saponin, which we named Lessonioside C. Further, the consecutive losses of the deacetylated aglycone, MeGlc, Glc, Xyl (at a terminal position) and an acetyl group from the parent ion generated the fragment ions at *m/z* 981.3, 805.3, 643.2, 493.1 and 433.1, (Figure 3d) respectively, confirming the structure of Lessonioside C.

Secondly, the decomposition of the parent ion can also be triggered by the sequential loss of sugar moiety namely MeGlc, Xyl, Glc, AcOH, MeGlc, Qui and Xyl followed by the deacetylated aglycone residue which generated daughter ions at *m/z* 1301.6, 1169.6, 1007.5, 947.5, 771.4, 625.2, and 493.1, respectively (Figure 3e). This sequence of fragmentation confirms the structure of new saponin, Lessonioside A. In this case, the ions at *m/z* 493.1 correspond to the sodiated deacetylated aglycone moiety (*m/z* value of 470).

The third viable pathway is elicited by the initial loss of an acetoxy group. In the case of Lessonioside A this initial loss (−60) is followed by the sequential loss of the sugars (including the diagnostic MeGlc-Glc-Xyl) to yield the key diagnostic DeAc Agl ion (*m/z* 493.1) (Figure 3f). In addition the sequential losses of the MeGlc (317.1) and Xyl (*m/z* 185.0) followed by Glc further confirmed the structure of the new isomer, Lessonioside A.

As was observed with the MALDI-MS<sup>2</sup> this saponin possesses the common *m/z* 493.1 key signal diagnostic of both the sugar moiety [MeGlc-Glc-Xyl + Na]+ and the DeAc Agl moiety [C32H50O6 − AcOH + Na]+. This is consistent with previous findings for the MS2 of sea cucumber saponins [12]. The ion 493.1 is also observed in Lessonioside C, however, this is formed as a result of a loss of DeAc Agl and of the sugar moiety MeGlc-Glc-Xyl (511) and H2O (493). The ion at 643 yields ions at 511 and 493 by the loss of Xyl and Xyl + H2O, respectively. These ions are recognized as the key diagnostic fragments in triterpenoid saponins.

These MS<sup>2</sup> analyses using both MALDI and ESI modes allowed the establishment of connectivities of the sugar residues and thus permit the assignment of the peaks. For example, the MALDI-MS2 of the parent ion showed fragments at *m/z* 1417.7 [M + Na − AcOH]+ which suggested the presence of an acetyl moiety and the innate sugar component at *m/z* 947.5 [M + Na − Agl]+, an observation that was confirmed by ESI- MS2 in the positive ion mode. The MALDI mass spectrum showed evidence of the presence of the acetoxy group in the aglycone.
