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.