Table 3. Detection limit and sensitivity improvement of phosphopeptides using the optimized 3-AQ/CHCA method and the conventional 2,5-DHB method.a) [Reproduced from ref. 13 with Copyright permission of John Wiley and Sons.].
| Phosphopeptides | Detection limit (fmol/well) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| [M+H]+ | ||||||||||
| 3-AQ/CHCA | 2,5-DHB | Improvement by 3-AQ/CHCA to 2,5-DHB-(3) (fold) | ||||||||
| No.b) | BB Indexc) | Phosphosites | [M+H]+ m/z | Focus plate (NH4)3PO4 | (1) SUS plate no additive | (2) Focus plate no additive | (3) Focus plate (NH4)3PO4 | |||
| 1 | β-Casein 33–48 | Isolated | +6200 | 1 | 2061.8 | 0.1 | 1 | 1 | 1 | 10 |
| 2 | β-Casein 1–25 | Isolated | −120 | 4 | 3122.3 | 0.1 | 1000 | 10 | 1 | 10 |
| 1* | β-Casein 33–48 | Digests | +6200 | 1 | 2061.8 | 1 | 1 | 1 | 1 | 1 |
| 2* | β-Casein 1–25 | Digests | −120 | 4 | 3122.3 | 1 | 100 | 10 | 1 | 1 |
| 3 | Ovalbumin 341–360 | Digests | +4230 | 1 | 2088.9 | 0.1 | 10 | 1 | 1 | 10 |
| 4 | Ovalbumin 60–85 | Digests | +1430 | 1 | 2901.3 | 0.1 | 10 | 10 | 1 | 10 |
| 5 | IRK-3 | Isolated | −1590 | 1 | 1702.8 | 1 | 1 | 1 | 1 | 1 |
| 6 | IRK-1 | Isolated | −1590 | 3 | 1862.8 | 1 | 10 | 10 | 1 | 1 |
a) The detection limit of phosphopeptides was evaluated as described in Experiment section in ref. 13. Sensitivity improvement is determined by dividing the detection limit using the conventional method-(1), -(2), or -(3) with 2,5-DHB by that using the optimized method with 3-AQ/CHCA. b) Peptide-1* and -2* denote peptide-1 and -2 in digests, respectively. c) The BB Index of phosphopeptides is calculated by changing the phosphoserine residues to glutamic acid.34,35)