Protocol

Analysis of Phosphopeptides by µLC-ESI-MS/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

Whether using a pressure cell or gel-loader tips, IMAC-purified peptides can be injected into a µLC separation system and analyzed by µLC-ESI-MS/MS. Phosphopeptides behave and elute similarly to nonphosphorylated peptides during RP-HPLC, but because of a decrease in hydrophobicity due to the addition of the phosphate moiety, a phosphopeptide will generally elute before the nonphosphopeptide with the identical amino acid sequence. This difference in elution time is most pronounced for short peptides. Thus, when analyzing phosphopeptides from µLC online with ESI-MS/MS, it is prudent to acquire data during the wash step (i.e., prior to gradient elution), as some short phosphopeptides may not even bind to the C₁₈ resin in 100% aqueous solutions. Otherwise, these very hydrophilic peptides will be missed. This protocol provides typical µLC conditions for the separation of IMAC-enriched phosphopeptides when the µLC system is online with the ESI-MS/MS.