Protocol

Characterization of Phosphopeptides Using a Combination of Immobilized Metal Ion Affinity Media and Direct Analysis by MALDI-TOF-MS

This protocol was adapted from "Proteomic Methods for Phosphorylation Site Mapping," Chapter 9, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Phosphoproteins and peptides can be bound with high specificity to immobilized metal ions such as Fe³⁺, Ni²⁺, and Ga³⁺. 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 Fe³⁺ or Ga³⁺ metal IMAC with the on-bead analysis of phosphopeptides by MALDI-MS. Enzymatic digestion of affinity-bound peptides is also described.