Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway

Albrecht Gruhler,Jesper V Olsen,Shabaz Mohammed,Peter Mortensen,Nils J Færgeman,Matthias Mann,Ole N Jensen

doi:10.1074/mcp.m400219-mcp200

Abstract

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [(13)C(6)]arginine and [(13)C(6)]lysine in a double auxotroph yeast strain. Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control and lysed, and extracted proteins were digested with trypsin. Phosphopeptides were enriched by a combination of strong cation exchange chromatography and IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle.

Highlights

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation
In the present study we further explored this insight by combining strong cation exchange chromatography (SCX)1 with IMAC and LC-MS in a quantitative phosphoproteomic study of the yeast pheromone response
Generation of Yeast Strains and SILAC Labeling with [ 13C6 ]Arginine and [ 13C6 ]Lysine—Cellular protein expression levels and regulation of protein activity mediated by posttranslational modifications vary greatly in response to developmental or environmental changes, and quantitation of protein expression is an important aspect of proteomic studies

Summary

Introduction

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. One of the most important and best characterized post-translational modifications, plays a key role in eukaryotic signal transduction, gene regulation, and metabolic control in cells [3] Fundamental processes such as cell proliferation, adaptation, and differentiation are governed by reversible phosphorylation at specific serine, threonine, and tyrosine residues in proteins, and protein kinases and phosphatases are becoming major drug targets for a wide variety of diseases [4].

Objectives

Results

Conclusion