Table 3. AAV2 Viral Protein Ratios Determined by HILIC-FLR-MS at Column Temperatures of 25, 45, and 60 °C.
| column temp (°C) | FLRa | MSb | |||||||
|---|---|---|---|---|---|---|---|---|---|
| viral protein (VP) | average area (counts × min) | area CV (%) | VP copies | ratio | viral protein (VP) | average area (counts × min) | VP copies | ratio | |
| 25 | VP1 | 3541041.67 | 1.28 | 7.99 | 1.60 | VP1 | 81,572 | 6.79 | 1.36 |
| VP2 | 2557869.33 | 4.39 | 5.77 | 1.15 | VP2 | 57,274 | 4.77 | 0.95 | |
| VP3 | 19850718.67 | 0.80 | 44.77 | 8.95 | VP3 | 582,232 | 48.45 | 9.69 | |
| VP3 prime | 656673.33 | 9.80 | 1.48 | 0.30 | VP3 prime | ||||
| 45 | VP1 | 2801034.67 | 8.01 | 6.75 | 1.35 | VP1 | 105,131 | 7.07 | 1.41 |
| VP2 | 2762226.00 | 4.24 | 6.65 | 1.33 | VP2 | 52,038 | 3.50 | 0.70 | |
| VP3 | 18581684.00 | 1.35 | 44.77 | 8.95 | VP3 | 735,638 | 49.44 | 9.89 | |
| VP3 prime | 760355.00 | 11.57 | 1.83 | 0.37 | VP3 prime | ||||
| 60 | VP1 | 1885809.00 | 3.26 | 5.08 | 1.02 | VP1 | 79,079 | 6.99 | 1.40 |
| VP2 | 2907691.67 | 2.49 | 7.84 | 1.57 | VP2 | 35,040 | 3.10 | 0.62 | |
| VP3 | 17013525.67 | 2.70 | 45.85 | 9.17 | VP3 | 565,115 | 49.92 | 9.98 | |
| VP3 prime | 459231.67 | 28.61 | 1.24 | 0.25 | VP3 prime | ||||
The average areas were calculated using FLR traces with values calculated as an average of the three runs performed at each column temperature. Given that an AAV capsid contains a total of 60 VP copies, the number of VP copies per VP was determined by dividing the total number of VPs by the summed integrated area of VP1, VP2, VP3, and VP3 Prime and then multiplying the result by the area of each respective VP. VP ratios were calculated based on the theoretical ratio of VP1:VP2:VP3 being 1:1:10 as described in the literature. The summed total of 12 theoretical copies was divided by the summed integrated area of VP1, VP2, VP3, and VP3 Prime and then multiplied by the area of each respective VP. Complete calculations can be found in Table S7 of the Supporting Information.
The average areas were calculated using the area under the XICs of each proteoform identified during MS deconvolution. The areas of all proteoforms of a single VP were summed to represent the VP. An average of these sums across the three runs performed at each column temperature was used to calculate the total average area of each VP. VP copies and VP ratios were then calculated as described for the FLR method. Components in VP3 prime were minimal and thus were not included in this calculation. Complete calculations can be found in Table S8 of the Supporting Information.