Characterization of Phosphopeptides Using a Combination of Immobilized Metal Ion Affinity Media and Direct Analysis...

INTRODUCTIONPhosphoproteins and peptides can be bound with high specificity to immobilized metal ions such as Fe(3+), Ni(2+), and Ga(3+). This technique can be used with either on-line or off-line MS analysis. However, elution of the phosphopeptides from the metal ion column prior to MS analysis can result in sample loss. Affinity-bound analytes, including phosphopeptides, can be directly analyzed by MALDI-MS without prior elution from the affinity media. Also, consecutive enzymatic reactions, such as phosphatase or carboxypeptidase Y digestion, can be carried out on affinity-bound peptides. When the affinity-bound phosphopeptides are treated with phosphatase, the number of phosphorylation sites can be determined based on the observation of 80-Da (or multiples of 80 Da) mass shifts in the MALDI-MS of the reaction mixture. Carboxypeptidase Y treatment of the affinity-bound phosphopeptides can also be used to cleave amino acids from the carboxyl terminus, with subsequent direct analysis of the enzymatic products by MALDI-MS to locate the phosphorylation sites on the bound phosphopeptides. This protocol details the preparation and use of Fe(3+) or Ga(3+) metal IMAC with the on-bead analysis of phosphopeptides by MALDI-MS. Enzymatic digestion of affinity-bound peptides is also described.