Protocol

Histidine Phosphorylation Site Identification by ESI-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

Histidine phosphate is moderately stable under alkaline conditions; however, the half-life of this phosphoamino acid under acidic conditions (pH <5) is less than 20 min. Thus, it is better to process proteins containing phosphohistidine rapidly and under alkaline conditions. The use of acidic matrices such as -cyano-4-hydroxycinnamic acid for MALDI-MS prevents the detection of phosphohistidine. An alkaline MALDI matrix is a poor alternative because it often decreases the sensitivity of detection of phosphorylated amino acids. Surprisingly, phosphohistidine is stable under acidic conditions when stored on C₁₈ column resins. Therefore, HPLC separation on C₁₈ resin with an acidic solvent system combined with ESI-MS is possible.