Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway*S

By A. Gruhler, Jesper V. Olsen, S. Mohammed, P. Mortensen, Nils J. Færgeman, Matthias Mann, Ole Nørregaard Jensen. Molecular & Cellular Proteomics, 2005

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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 [13C6]arginine and [13C6]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.

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/cb9df3318f2096a695b0482ce827889794f6a879

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Citations in the corpus

The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry (2008, methodology): “However, the method that has proven most comprehensive in mapping the phosphoproteome is SCX chromatography combined with either IMA”
Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. (2006, methodology): “Protein digestion was performed essentially as in Foster et al. (2003), strong-cation exchange chromatography of digests as in Gruhler et al. (2005), and TiO2 enrichment of phosphopeptides essentially as in Larsen et al. (2005).”
Quantitative Phosphoproteomics of Early Elicitor Signaling in Arabidopsis*S (2007, methodology): “Recently MS(3) has been used to good effect to identify phosphorylated peptides in yeast (26).”
Structural Characterization of the Human Eukaryotic Initiation Factor 3 Protein Complex by Mass Spectrometry*S (2007, background): “Jensen and co-workers (48) have applied the same strategy on a hybrid linear ion trap-FTICR mass spectrometer that has the advantage of faster scan speeds, accurate mass measurements of precursor ions, and better storage of precursor ions.”
Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns* (2005, result): “(14) support this finding as they observed a significantly lower amount of multiphosphorylated peptides compared with singly”
Mass spectrometry–based proteomics turns quantitative (2005, methodology): “If a microorganism is amenable to genetic manipulation, however, a general method for SILAC labeling is the generation of auxotrophic strains, such as Arg − /Lys − double auxotrophic yeas”
Ultrasensitive proteome analysis using paramagnetic bead technology (2014, background): “In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential.”
Decoding signalling networks by mass spectrometry-based proteomics (2010, background): “An unbiased phosphoproteomics study on the pheromone pathway in yeast has quantified more than 700 phosphorylated peptide”
Proteomics by mass spectrometry: approaches, advances, and applications. (2009, positive): “Numerous studies applied SILAC to studying dynamic changes in response to stimuli (182, 183).”