Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Source induced dissociation (SID) with target MS2 for high throughput nanoLC-ESI-MS/MS mapping of multiple glycotopes (#574)

Cheng-Te Hsiao 1 , Po-Wei Wang 1 , Hua-Chien Chang 1 , Kay-Hooi Khoo 1
  1. Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan

Protein glycosylation affects and modulates the structure and functions of membrane and secreted proteins. The glycans are attached to either asparagine or serine/threonine via a few common core structures, which can be extended either in linear or branched form before terminally capped by sialic acid, fucose, and/or sulfate at different positions to constitute the critical terminal Glyco-epitopes, or glycotopes. We have recently developed an advanced glycomic workflow that homes in on identifying these glycotopes by comprehensive nanoLC-MS2-product dependent(pd)-MS3 analysis of permethylated glycans. The coupled MS3 step is required to distinguish the various isomeric variants of each glycotope, as defined by a specific MS2 diagnostic ion, via the further induced linkage-specific cleavages. We now introduce an alternative implementation of similar glycotope mapping by coupling source induced dissociation (SID) with target MS2 at tailored collision energy (CE), which also simultaneously allows selective data dependent MS2 acquisition of sodiated species at higher CE setting. Within a 3 sec top speed mode cycle, a full MS survey scan acquired in the Orbitrap is followed, as first priority, by target MS2 of a list of anticipated source induced fragment ions of interest, akin to a pseudoMS3. A 2nd priority HCD-MS2 acquisition at 50% NCE was then triggered on the sodiated form whenever a delta mass difference of 21.98 Da is detected to provide complementary sequence-informative fragmentation. A comparison with previously acquired pd-MS3 data showed that current target MS2 analysis produced similar results for high level glycomic mapping of the expressed glycotopes, including ± sialyl Lex/a, Ley/b, and disialylated Hex-3/4HexNAc glycotopes. Importantly, it yielded much better quality MS2 spectra, instead of otherwise required pd-MS3, to allow higher confident ID and relative quantification, as well as being able to target more glycotopes to be verified at MS2 level in a single run.