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2D-LC analysis of phosphopeptides in brain tissue using Ettan MDLC and Finnigan LTQ

Key words: phosphopeptide • two-dimensional liquid chromatography (2D-LC) • Ettan MDLC • LC-MS/MS • reversed-phase chromatography (RPC) • strong cation exchange (SCX) chromatography • linear ion trap mass spectrometer


A 2D–LC-MS method was developed to analyze phospho-peptides in mouse brain tissue. The trypsin-digested tissue was separated by strong cation exchange chromatography (SCX), followed by reversed-phase chromatography (RPC) using Ettan™ MDLC. The detection was performed by mass spectrometry using neutral loss of phosphoric acid to selectively detect the phosphorylated peptides. Several phosphorylation sites were found, and a strategy for confident assignment of these was developed.


Introduction
One of the most important post-translational modifications is phosphorylation of serine, threonine, or tyrosine residues. Phosphorylated proteins play important roles in a wide range of biological processes, such as signal transduction, apoptosis, and cell cycle control. Detection of phosphorylation sites by mass spectrometry in proteins extracted from biological material is hampered by the low abundance, low stoichiometry, and poor ionization of phosphopeptides (1).

In this work, a biocompatible nanoscale liquid chromatography (LC) system, Ettan MDLC, was used for separating phosphopeptides. No metal ions that can chelate phosphate groups are present in the fluid pathway of the LC system, resulting in highly sensitive analyses (2).

Separation of the tryptic peptides was performed in two dimensions, SCX followed by RPC. A Finnigan™ LTQ™ linear ion trap mass spectrometer was used for detecting phosphopeptides by fragmenting all peptides that exhibited a neutral loss of phosphoric acid.


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