Table 4.
De novo sequencing of ‘NOX-E36 Int. mutant’ (Table 1) readily reveals both CU mutations compared to the parent NOX-E36 Int. Spiegelmer
| Fragment # | RT in TICa (min) | Type of Observable mass | Deconvoluted Observed mass (Da) | ΔMassb (Da) | ΔMass identified asc | Calculated massd |
Absolute errore (Da) | |
|---|---|---|---|---|---|---|---|---|
| Exact (Da) | Average (Da) | |||||||
| 1 | 3.87 | Exact | 612.12 | 612.12 | G | 612.12 | – | 0.00 |
| 2 | 5.17 | Exact | 917.16 | 305.04 | C | 917.16 | – | 0.00 |
| 3 | 9.02 | Exact | 1246.21 | 329.05 | A | 1246.21 | – | 0.00 |
| 4 | 11.84 | Exact | 1551.25 | 305.04 | C | 1551.25 | – | 0.00 |
| 5 | 14.84 | Exact | 1896.30 | 345.05 | G | 1896.30 | – | 0.00 |
| 6 | 17.25 | Exact | 2202.32 | 306.02 | U | 2202.32 | – | 0.00 |
| 7 | 19.14 | Exact | 2507.36 | 305.05 | C | 2507.37 | – | 0.01 |
| 8 | 20.73 | Exact | 2812.40 | 305.03 | C | 2812.41 | – | –0.01 |
| 9 | 22.08 | Exact | 3117.44 | 305.03 | C | 3117.45 | – | –0.01 |
| 10 | 23.21 | Exact | 3422.48 | 305.03 | C | 3422.49 | – | –0.01 |
| 11 | 24.26 | Exact | 3728.51 | 306.02 | U | 3728.52 | – | −0.01 |
| 12 | 25.65 | Exact | 4057.56 | 329.04 | A | 4057.57 | – | –0.01 |
| 13 | 26.29 | Exact | 4362.60 | 305.03 | C | 4362.61 | – | –0.01 |
| 14 | 26.98 | Exact | 4667.64 | 305.03 | C | 4667.65 | – | –0.01 |
| 15 | 27.87 | Exact | 5012.68 | 345.03 | G | 5012.70 | – | –0.02 |
| 16 | 28.76 | Exact | 5357.74 | 345.04 | G | 5357.75 | – | –0.01 |
| 17 | 29.61 | Exact | 5663.75 | 306.00 | U | 5663.77 | – | –0.02 |
| 18 | 30.59 | Exact | 6008.80 | 345.03 | G | 6008.82 | – | –0.02 |
| 19 | 31.47 | Exact | 6313.86 | 305.04 | C | 6313.86 | – | 0.00 |
| 20 | 33.23 | Exact | 6642.92 | 329.06 | A | 6642.91 | – | +0.01 |
| 21 | 34.84 | Exact | 6971.96 | 329.05 | A | 6971.96 | – | 0.00 |
| 22 | 35.73 | Exact | 7316.99 | 345.03 | G | 7317.01 | – | –0.02 |
| 23 | 36.58 | Exact | 7623.02 | 306.01 | U | 7623.04 | – | –0.02 |
| 24 | 37.62 | Exact | 7968.07 | 345.03 | G | 7968.08 | – | –0.01 |
| 25 | 39.32 | Exact | 8297.13 | 329.05 | A | 8297.14 | – | –0.01 |
| 26 | 40.88 | Exact | 8626.23 | 329.09 | A | 8626.19 | – | +0.04 |
| 27 | 41.56 | Exact | 8971.30 | 345.11 | G | 8971.24 | – | +0.06 |
| 28 | 42.22 | Exact | 9276.28 | 305.04 | C | 9276.28 | – | 0.00 |
| 29 | 42.95 | Exact | 9581.26 | 304.98 | C | 9581.32 | – | –0.06 |
| 30 | 43.84 | Exact | 9926.35 | 345.03 | G | 9926.37 | 9931.02 | –0.02 |
| 31 | 44.54 | Average | 10237.26 | 306.24 | U | – | 10237.19 | +0.07 |
| 32 | 45.31 | Average | 10582.53 | 345.34 | G | – | 10582.40 | +0.13 |
| 33 | 46.09 | Average | 10927.69 | 345.29 | G | – | 10927.60 | +0.09 |
| 34 | 46.66 | Average | 11233.75 | 306.15 | U | – | 11233.77 | –0.02 |
| 35 | 47.21 | Average | 11538.95 | 305.18 | C | – | 11538.95 | 0.00 |
| 36 | 47.76 | Average | 11844.25 | 305.30 | C | – | 11844.13 | +0.12 |
| 37 | 48.28 | Average | 12150.34 | 306.21 | U | – | 12150.29 | +0.05 |
| 38 | 48.58* | Average | 12495.64 | 345.35 | G | – | 12495.50 | +0.14 |
| 39 | 49.26** | Average | 12800.80 | 305.30 | C | – | 12800.68 | +0.12 |
| FLP | 48.58 | Average | 13084.04 | 283.36 | G | – | 13083.92 | +0.12 |
aRetention time values are those taken from the TIC.
bΔ mass is calculated by subtracting the calculated mass value of the previous fragment from the observed value.
cSee Table 3 for cipher.
dCalculated mass values are obtained by calculating the theoretical mass of the confirmed sequence.
eAbsolute error is calculated by subtracting the target Δ mass value (Table 3) from the Δ mass value obtained.
*Fragment 38 co-elutes with the full-length (undegraded) released product (FLP). **Fragment 39 has a retention time greater than the FLP, which in this case is HS-(CH2)2C(O)-NH(CH2)6-OP(O)(OH)-GCACGUCCCCUACCGGUGCAAGUGAAGCCGUGGUCCUGCG.
The C/U switches compared to ‘NOX-E36 Int.’ (Table 1) are highlighted (bold).