Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Summary

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Abstract

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis. Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics. Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Protocol

Cell growth and media

1. A single colony of BY4742Δarg4Δlys1 cells overnight in 100 mL rich media to an OD₆₀₀ of 1.0 then seed into two 1 liter cultures of a minimal yeast medium (0.17% Yeast Nitrogen Base without Ammonium Sulfate or Amino Acids, 0.5% Ammonium sulfate) containing a full complement of amino acids, supplemented with 20mg/L of isotopically normal or heavy arginine (¹³C₆ ¹⁵N₄; Isotec) and lysine (¹³C₆ ¹⁵N₂; Isotec).
2. The cells were grown for 18 hours, to an OD₆₀₀ of 1.8. It is critical that the cells....

Discussion

This method will yield an enrichment of phosphopeptides that can be quantitatively analyzed. A number of parameters can be altered in this protocol, but the most important aspect to remember is to preserve your phosphorylation. Preparation of cells for quantification can be achieved in a number of different ways, for example if you cannot label your cells in vivo, tags such as ICAT[3] or ITRAC [4] can be used post extraction to differentially label two cultures for quantification purposes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isotec™ L-Arginine-13C6,15N4	Sigma-Aldrich	608033
Isotec™ L-Lysine-15N2	Sigma-Aldrich	609021
GIBCO™ Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid	Invitrogen	70011044
SigmaFAST Protease Inhibitor Tablets	Sigma-Aldrich	S8820
Pierce Halt Phosphatase Inhibitor Cocktail	Thermo Fisher Scientific, Inc.	78420
Retsch PM100 Ball Mill Grinder	Retsch	20.540.0001
Retsch 125 ml Stainless Steel Grinding Jar	Retsch	01.462.0148
Ultramicrospin C18 column	The Nest Group	SUM SS10
Sep-Pak Vac 500 mg C18	Waters	WAT043395
TSK-Gel Amide-80 4.6 mm x 25 cm analytical column	Tosoh Corp.	13071	This is the HILIC chromatographic column.
Guard column 4.6 mm ID x1 cm	Tosoh Corp.	19021	We recommend this guard column to extend the life of your HILIC column.
PHOS-Select™ Iron Affinity Gel	Sigma-Aldrich	P9740	This is the IMAC resin. Aliquot and store.