( ...)Quantification of the Purinergic Receptor P2X3 using ICAT and ,,, Orthogonal 2D LC-MS/MS with an Ion Trap Mass Spectrometer

David Barnidge, Leo E. Bonilla, Gargi Choudhary

Neuromics, Inc., Minneapolis, MN
Thermo Finnigan, San Jose, CA

ICAT Analysis

The data presented here was acquired using the ProteomeX integrated proteomics workstation. Overview The differential quantification of the purinergic receptor P2X3 in membrane preparations of kidney cells is demonstrated using a fully integrated 2D LC-MS/MS system to detect and quantify ICAT-labeled tryptic peptides. Optimization of ICAT procedures, by removal of the need for off-line ion exchange (IEX) and affinity clean-up after ICAT derivatization, is demonstrated. Introduction The systematic identification and quantification of the constitutive proteins in a complex system (e.g., organelle, cell, tissue) is the central task of proteomics. Orthogonal two-dimensional chromatography (2D-LC) coupled with tandem mass spectrometry (MSⁿ) represents a powerful technology platform for the global study of proteomes.⁽¹⁾ Stable-isotope labeling, such as isotope-coded affinity tags (ICAT), allows the implementation of MS-based methods for quantitative protein profiling.

The ICAT procedure relies on the selective alkylation of the free thiol groups of cysteines with either a d₀- or d₈- ICAT followed by proteolysis, affinity enrichment of the labeled peptides, and MS analysis.⁽²⁾ Quantitative ICAT labeling of membrane proteins remains an imp
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