Phosphorylation

Elution Selectivity Maldi Ms

Product No: P 1617

Plate Elution Selectivity - MALDI-MS

Figure 4. MALDI mass spectra of A) Crude ß-casein tryptic digest (4.5 pmol/5L) prior to loading on the PHOS-Select Plate and B)-E) purified phosphopeptides after elution from the PHOS-Select Plate under various elution conditions. Eluents are as described in Figure 2.

Binding Capacity Study
Species Molecular
Weight (Da)
Format
Plate
(nmol/well)
Resin
(µmol/mL)
4-methylumbelliferyl
phosphate (MUP)
256.14 0.9 4.5
Monophosphopeptide
(ß-casein)
2061.96 0.5 8.0
Tetraphosphopeptide
(ß-casein)
3122.92 1.0 5.5
Binding capacities for the two formats were determined using purified phosphorylated molecules. Binding of MUP was determined via a fluorescence assay. Binding of the mono and tetraphosphopeptides was determined using HPLC-MS analysis.

Table 1 Elution Conditions
Elution Solutions Comments
0.2 M sodium phosphate, pH 8.4 Provides competitive displacement. Requires salt removal for MALDI-TOF-MS.
0.15 to 0.4 M ammonium hydroxide Provides significant recovery of phosphocompounds. Compatible with MALDI-TOF-MS.
0.15 M ammonium hydroxide with 25% acetonitrile Provides significant recovery of phosphocompounds. Compatible with MALDI-TOF-MS.
1 to 5% TFA in water Provides significant recovery of monophosphocompounds. Compatible with MALDI-TOF-MS.