Glycoprotein CCS Values
To support data interpretation of IM and MS/MS data we will populate a new CCS database, GlycoMob, as well as supplement the existing glycan fragmentation database UniCarb-DB with measurements of glycan standards. Glycan analysis using IM-MS is a relatively new approach and applications for CCS measurements have principally employed home-built instruments. As a consequence the wide-spread use of IM-MS for glycomics analysis on commercially available instruments has been hindered.
Glycoprotein Standards Summary
| Glycoprotein | Positive Mode using Helium | Positive Mode using Nitrogen | Negative Mode using Helium | Negative Mode using Nitrogen |
|---|---|---|---|---|
| Thyroglobulin | 35 (69) | 35 (69) | - | - |
| Fetuin | 7 (24) | 7 (24) | - | - |
| Fetuin Ds | 12 (37) | 12 (37) | 12 (19) | 12 (19) |
| Rnase B | 8 (34) | 8 (34) | 8 (42) | 8 (42) |
| Thyroglobulin Ds | 35 (69) | 35 (69) | 13 (54) | 13 (54) |
| Ovalbumin | 24 (51) | 24 (51) | 21 (105) | 21 (105) |
*Content in parenthesis refers to the total number of native and fragment entries. Values outside parenthesis correspond to the number of native structures
Ion Mobility
Method Summary
Measurements of absolute CCS values were performed using a Synapt G1 HDMS quadrupole/IMS/oa-ToF instrument (Waters Co., Manchester, U.K.) modified for drift tube operation (Bush, et al., 2010). Briefly, N-linked glycans were released with hydrazine from the well-characterized glycoproteins ribonuclease B, porcine thyroglobulin, chicken ovalbumin, and bovine fetuin obtained from Sigma Chemical Co., Ltd. (Poole, Dorset, U.K.) and reacetylated. Sialic acids were removed from the thyroglobulin and fetuin samples by heating with 1% acetic acid for 1 h at 70 °C. For electrospray analysis, samples were dissolved in water:methanol (1:1, v:v) at ∼1 mg/mL. More information refer to the supplementary section Pagel K and Harvey DJ, Analytical Chemistry 2013.