Table 2.
Amino acid sequence of the proposed fluorescein-labeled peptides Fluo-I-III, their acetylated homologs, Ac-I-III, and binding constant determination for the fluorescein-labeled.
| Entry | Peptide | Peptide Sequence | tR a (min) | Mass d | Binding Constant Values | ||
|---|---|---|---|---|---|---|---|
| Calculated | Found | KD (nM) e | ΔmP | ||||
| 1 | Fluo-Ia | Fluo-LNDNHEELIQ | 19.7 b | 1582.6332 | 1582.6327 | 74.5 ± 10.5 | 127 |
| 2 | Fluo-Ib | Fluo-FENRLNDNHEELIQ | 17.4 b | 2128.8883 | 2128.8901 | 89.5 ± 13.9 | 105 |
| 3 | Fluo-Ic | Fluo-TRKQFENRLNDNHEELIQ | 17.2 b | 2642.1906 | 2642.1822 | No binding | - |
| 4 | Fluo-IIa | Fluo-LRSAEFEETQ | 20.1 b | 1567.6223 | 1567.6235 | 169.5 ± 16.0 | 61 |
| 5 | Fluo-IIb | Fluo-GDTSLRSAEFEETQ | 18.1 b | 1927.7505 | 1927.7498 | 99.5 ± 12.2 | 110 |
| 6 | Fluo-IIc | Fluo-GSGDTSLRSAEFEETQ | 18.3 b | 2071.8040 | 2071.8009 | 134.0 ± 15.0 | 106 |
| 7 | Fluo-IIIa | Fluo-ERYKTMYADD | 17.4 b | 1649.6213 | 1650.2056 | No binding | - |
| 8 | Fluo-IIIb | Fluo-HGTIERYKTMYADD | 16.2 b | 2057.8334 | 2057.0183 | No binding | - |
| 9 | Fluo-IIIc | Fluo-INSYHGTIERYKTMYADD | 18.4 b | 2536.0398 | 2536.1358 | No binding | - |
| 10 | Ac-Ia | Ac-LNDNHEELIQ | 14.3 c | 1266.5961 | 1266.5998 | n/a | n/a |
| 11 | Ac-Ib | Ac-FENRLNDNHEELIQ | 14.0 c | 1812.8511 | 1812.8543 | n/a | n/a |
| 12 | Ac-Ic | Ac-TRKQFENRLNDNHEELIQ | 13.2 c | 2325.1682 | 2325.1653 | n/a | n/a |
| 13 | Ac-IIa | Ac-LRSAEFEETQ | 15.8 c | 1251.5852 | 1251.5811 | n/a | n/a |
| 14 | Ac-IIb | Ac-GDTSLRSAEFEETQ | 13.8 c | 1611.7133 | 1611.7113 | n/a | n/a |
| 15 | Ac-IIc | Ac-GSGDTSLRSAEFEETQ | 13.5 c | 1755.7668 | 1755.7641 | n/a | n/a |
| 16 | Ac-IIIa | Ac-ERYKTMYADD | 13.4 c | 1333.5841 | 1333.5891 | n/a | n/a |
| 17 | Fluo-Id | Fluo- LNANHEELIQ | 18.5 c | 1538.6434 | 1538.6474 | No binding | - |
| 18 | Fluo-Ie | Fluo-DNLNHEELIQ | 18.3 c | 1582.6332 | 1582.6327 | 175.0 ± 8.90 | 115 |
| 19 | Fluo-If | Fluo-NDNLEE | 17.5 c | 1091.3476 | 1091.3465 | 74.4 ± 8.05 | 199 |
| 20 | Fluo-Ig | Fluo-DNLEE | 17.5 c | 976.2974 | 976.2969 | 71.6 ± 9.15 | 225 |
a HPLC purification using Agilent Technologies (Santa Clara, CA, USA) 1260 Infinity equipment, a reverse phase column (Nucleodur, C-18 HTec, 5 µm, Macherey-Nagel, Düren, Germany), and a solvent mixture of water and acetonitrile containing 0.01% TFA. The purity % (UV, λ = 210 nm), for all peptides was 100%; b Purification was performed starting from 25% B to 95% B over 40 min at a flow rate of 30 mL·min−1 with a linear gradient; c Purification was performed starting from 5% B to 95% B over 40 min at a flow rate of 30 mL·min−1 with a linear gradient; d nLC-MS/MS ESI (electrospray ionization) interface (Advion). Fluo: N-5-(6)-fluoresceinyl-carboxy-; Ac: acetyl group; e Values represent three independent experiments, including three replicates (n = 3 ± SD); n/a: not applicable.