Unveiling Phosphorylation Modification Using Phos-tag SDS-PAGE Gel Electrophoresis and In Vitro Kinase Assay

Summary

This protocol describes procedures to detect changes in phosphorylation modifications of target proteins using Phos-tag gel electrophoresis in clinical samples and in vitro kinase assays.

Abstract

Phosphorylation is a classic post-translational modification that regulates protein function. Proteins typically contain multiple potential phosphorylation sites, which can be modified by various kinases at different locations. Studying phosphorylation changes of target proteins in disease often requires phospho-specific antibodies. However, commercial options may be limited to a single site or entirely unavailable. Here, a method is described to detect changes in phosphorylation modifications of target proteins in clinical samples using the Phos-tag gel electrophoresis and to identify detailed phosphorylation sites through in vitro kinase assays. Phosphorylated proteins bound to Phos-tag exhibit slower migration rates in SDS-PAGE gel electrophoresis, enabling semi-quantitative analysis of phosphorylation changes in disease tissues based on the mean pixel intensity of the slowly migrating bands. By combining Phos-tag SDS-PAGE with immunoblotting using pan-specific antibodies against multiple candidate proteins, researchers can efficiently identify target proteins with phosphorylation changes. Following the screening of candidate kinases, in vitro kinase assays are performed with the target protein, and the resulting phosphorylated products are subjected to mass spectrometry for precise site identification. This method does not require specific phosphorylated protein antibodies, allowing large-scale screening of tissue samples to identify altered phosphorylation states in disease. Furthermore, the identified phosphorylation sites can be used to develop specific antibodies for quantitative and localization analysis in disease tissues, providing deeper insights into their functional roles.

Introduction

Phosphorylation is a key post-translational modification for modulating the subcellular localization, kinase activity, and transcriptional activity of proteins. More than 500 protein kinase genes have been found in the human genome, which are involved in signal transduction, gene expression, and protein-protein interaction¹. Phosphorylation and dephosphorylation are under the control of various kinases, which are quickly reversible to regulate cell adhesion, cell proliferation, and cell differentiation². However, abnormal protein phosphorylation modifications are responsible for many human diseases, including tumorigenes....

Protocol

The Institutional Review Boards at Nanjing Drum Tower Hospital approved the human research (2013-408081-01). The current protocol is established using endometrial tissue as a representative model. With appropriate optimization, this methodology can be extended to other clinical specimens, including but not limited to tumor tissues for protein extraction and subsequent analyses. Endometrial biopsy is performed in the mid-secretory phase from fertile women (Control group) and women with recurrent embryo implantation failure undergoingin vitro fertilization-embryo transfer (Disease group). All patients provided informed consent prior to the sampling procedure. T....

Results

The preliminary phosphoproteomic profiling of human samples from the control group and the disease group identified Nur77 as a candidate differentially phosphorylated protein. To evaluate the efficacy of Phos-tag SDS-PAGE in detecting Nur77 protein phosphorylation, a Mn²⁺-Phos-tag SDS-PAGE experiment was performed. After total protein extraction from tissue samples, 30 µg of protein was loaded and separated by Mn²⁺-Phos-tag gel electrophoresis, followed by transfer onto PVDF membrane. Immunoblotting.......

Discussion

The Phos-tag-based approach for detecting phosphorylation changes of target proteins in clinical tissues offers significant advantages for protein phosphorylation research. Phosphorylated proteins exhibit reduced electrophoretic mobility due to Phos-tag binding, enabling high-throughput screening of the phosphorylation status of candidate proteins without requiring phospho-specific antibodies. This is particularly valuable for initial large-scale profiling of multiple candidate proteins. Proteins with altered phosphoryla.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% SDS Solution	Sangon Biotech	B548118
10x Tris/Glycine/SDS	BIO-RAD	1610732
4×  Laemmli protein sample buffer	BIO-RAD	1610747
Acrylamide	Sangon Biotech	A601032
Ammonium persulfate	Sangon Biotech	A100486
Anti-FLAG M2 affinity gel	Millipore	A2220
Enhanced chemiluminescence kit	ThermoFisher	32106
FLAG antibody	CST	14793
FLAG Peptide	Millipore	F3290
HRP-conjugated secondary antibody	Bioworld Technology	BS13278
Lipofectamine 3000	ThermoFisher	L3000015
Mini Trans-Blot Cell	BIO-RAD	1703930
Mini-PROTEAN Tetra Vertical Electrophoresis Cell	BIO-RAD	1658005
Mst1 antibody	CST	3682
N, N'-methylene-bisacrylamide	Sangon Biotech	A600025
N,N,N',N'-Tetramethylethylenediamine(TEMED)	Sangon Biotech	A610508
Nur77 antibody	CST	3960
Phosphatase Inhibitor Cocktail 2	Sigma	P5726	For the inhibition of tyrosine protein phosphatases, acid and alkaline phosphatases
Phosphatase Inhibitor Cocktail 3	Sigma	P0044	For the inhibition of protein phosphatase 2A, alkaline phosphatases, protein phosphatases 1 and 2A
Phospho-serine antibody	Sigma	P5747
Phospho-threonine antibody	Sigma	P6623
Phos-tag Acrylamide	Wako	304-93526	For the preparation of Phos-tag SDS PAGE gel
Pierce BCA Protein Assay Kit	Thermo	23227
Protease Inhibitor Cocktail	Sigma	S8830	EDTA-Free, tablet, for the inhibition of serine, cysteine, aspartic and metalloproteases
PVDF membrane	Roche	3010040001
ThermoMixer	Eppendorf	2231001127
Tissue Disperser	IKA	T 10	To obtain tissue homogenate
Tris-HCl Buffer (0.5M, pH 6.8)	Sangon Biotech	B546020
Tris-HCl Buffer (1.5M, pH 8.8)	Sangon Biotech	B546019
Vacuum desiccator	ThermoFisher	5311-0250	To degas the gel solution
β-Mercaptoethanol	Sigma	M3148