SILAC-Based Quantitative Phosphoproteomics in Yeast

María Luisa Hernáez; Concha Gil

doi:10.1007/978-1-0716-2863-8_8

SILAC-Based Quantitative Phosphoproteomics in Yeast

Methods Mol Biol. 2023:2603:103-115. doi: 10.1007/978-1-0716-2863-8_8.

Authors

María Luisa Hernáez¹, Concha Gil^{2

3}

Affiliations

¹ Unidad de Proteómica, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain.
² Unidad de Proteómica, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain. conchagil@ucm.es.
³ Departamento de Microbiología y Parasitología. Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain. conchagil@ucm.es.

PMID: 36370273
DOI: 10.1007/978-1-0716-2863-8_8

Abstract

In this chapter, detailed procedures for stable isotope labeling with amino acids in cell culture, SILAC labeling of yeast auxotroph, optimization and evaluation of phosphopeptide enrichment, and sample preparation and analysis by high-resolution LC-MS/M, identification of phosphosites, and quantification methods are described.We report methods for the application of double SILAC to yeast using a combination of labeled lysine and labeled arginine.The combination of SILAC-based quantitation with phosphopeptides enrichment by TiO₂ in a batch that enables measurement of protein posttranslational modifications is a powerful application to analyze the global phosphoproteome for studies in signaling pathways.

Keywords: In-solution digestion; Phosphopeptides enrichment; Phosphoproteomics; Q-Exactive; Quantitative Proteomics; SILAC; TiO2.

MeSH terms

Isotope Labeling / methods
Mass Spectrometry / methods
Phosphopeptides / metabolism
Proteome / metabolism
Proteomics* / methods
Saccharomyces cerevisiae* / metabolism

Substances

Proteome
Phosphopeptides