Fig. 3.
NR-end Hn oligos created by OTH digestion are readily detected. Lifecore HA (542 kDa) was partially digested with OTH to a size of 49 kDa, to create ~11 times as many HA molecules with >90% having new A–G–A–G- NR-ends. The 49 kDa HA was purified by SEC, digested extensively with lyase and the final digest subjected to solid phase extraction as in Figure 2 and Methods. The indicated fractions were examined by MALDI-TOF MS in negative mode for m/z signals at 775 (H2), 1154 (H3), 1533 (H4) and 1912 (H5). (A) The four H2–5 species were not evident in digests of SeHAS HA. (B) In contrast, in the positive control-HA that had been predigested with OTH, all four H2–5 species were present in Wash 2 and H2–3 were found in Wash 3. Vertical dashed lines indicate positions where the four m/z species would be in samples with no signals for those species. Spuriously high G1Hn levels in lyase digests, first detected in this experiment, were confirmed in subsequent experiments (Table IV).