19
Table 3: Extract of a compound report.
extracted glycan signals against the database attaches corresponding structures to the hits. Figure 6 shows results obtained from the Antibody Standard. The identified hits are listed in the Data Navigator pane and corresponding peaks in Chromatogram Results (here, superposition of Extracted Compound Chromatograms). The Structure Viewer illustrates the glycan structure of the selected peak. m/z values, charge states and isotopes of the selected glycan hit are displayed in MS Spectrum Results and MS Spectrum Peak List.
Reporting: Volumes of glycosylamines with identical mass are merged. The same occurs for the volumes of the free reducing end glycan isomers. Latter are multiplied with an ionisation factor that compensates for the difference in ionisation efficiency between glycosylamines and free reducing end glycans. Corresponding glycosylamines and free glycans are clustered and the total volume is reported. Volumes of free reducing end glycan entries are deleted as redundant information. Finally, glycosylamine isomers are visualised by colour coding as shown in Table 3, which shows the summary table of a compound report that usually also includes chromatograms and mass spectra.
Data processing including reporting (all performed by a single mouse click) typically needs less than 5 minutes per sample. This makes a total of about 20 minutes for an assay that traditionally could take one half to several days.
Chip stability, reproducibility and lifetime Table 4 summarises characteristics of the mAb-Glyco Chip. Retention time stability over 200 injections was in the range 0.3 to 0.5% RSD (4 chips evaluated), reproducibility of the relative abundance was typically better
Figure 6: Typical results obtained from the extraction and identification procedure using MFE and glycan database search, respectively. Sample: Antibody Standard, 75ng on-column.
Investigated Item
Retention time stability over 200 injections
Intra-day reproducibility of rel. glycan ratios
Inter-day reproducibility of rel. glycan ratios
Spray stability Chip lifetime
PNGase F activity // long-term storage
PNGase F activity // freeze/thaw cycles
# of Chips tested 4 2 2 1 10 1 2 Table 4: Performance characteristics of the mAb-Glyco Chip. Results 0.3-0.5% RSD
5% RSD on average (glycans > 1% relative ration)
7% RSD on average (glycans > 1% relative ration)
> 200 hours 200-300 injections
(75ng lgG from bovine serum (Sigma) on-column each injection)
90% remaining activity after 3 months of storage at -20o
C
83% remaining activity after 74 freeze/thaw cycles
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