Table I.
Fractionation of phosphopeptides by stepwise elution from a titania columna
| Elution step | Condition A |
Condition B |
||
|---|---|---|---|---|
| Phosphate concentration (mM) and pH | Number of phosphopeptides detected | Phosphate concentration (mM) and pH | Number of phosphopeptides detected | |
| 1 | 5 (pH 2.3) | 686 (218)b | 5 (pH 2.3) | 638 (201)b |
| 2 | 50 (pH 2.6) | 785 (274)b | 20 (pH 2.4) | 689 (171)b |
| 3 | 200 (pH 3.5) | 395 (112)b | 50 (pH 2.6) | 439 (117)b |
| 4 | 500 (pH 8.0) | 179 (13)b | 500 (pH 8.0) | 444 (133)b |
| Total | 1,161 | 1,174 | ||
aA tryptic digest of a cytosol fraction of Xenopus eggs without a Ca2+-stimulus (4 mg) was applied to an automated phosphopeptide purification system. Phosphopeptides bound on a titania column were eluted stepwise with phosphate buffers, Conditions A or B. The obtained fractions were dried in vacuo and then desalted with ODS tips. The thus clarified phophopeptides derived from 1.2 mg of starting protein were dissolved in 0.1% formic acid-2% acetonitrile and then analysed by LC MS/MS.
bNumbers of phosphopeptides specifically detected in the fraction.