An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Summary

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Abstract

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

Introduction

Membrane-associated signaling is tuned by a combination of ligand-induced membrane receptor activation and recruitment of downstream accessory proteins that propagate the signal. Phosphorylation of key tyrosines in receptor cytoplasmic tails is critical to initiating the formation of signaling complexes, or signalosomes^1,2. Therefore, an important question in biology is how phosphorylation patterns are created and maintained to recruit signaling partners and dictate cellular outcomes. This includes understanding the heterogeneity of receptor phosphorylation, both in abundance and in the specific phosphotyrosin....

Protocol

1. Coverslip preparation

NOTE: For this step, one needs to wear personal protective equipment (PPE), which includes a double layer of nitrile gloves, safety glasses or face shield, and a lab coat.

1. Perform piranha etching to remove organic debris from the glass.
   CAUTION: Piranha solution is a strong oxidizing agent that is corrosive and highly reactive when in contact with organic materials. Reaction with organic debris is exothermic and potentially explosive. Thus, the procedure must be performed in a chemical fume hood with the sash lowered. Pyrex glassware is required to handle the piranha solution.

Results

A cartoon depicting the SiMPull process is shown in Figure 1A. Coverslips are functionalized using NeutrAvidin as an anchor for biotinylated anti-EGFR antibodies to capture EGFR-GFP from total protein lysates. After washing away unbound protein, the phosphorylated receptors are labeled with an anti-phosphotyrosine (anti-PY) antibody¹⁵. Figure 1B shows an image of the hydrophobic array, where multiple samples can be prepared and imaged on .......

Discussion

The protocol described here was optimized to enable quantitative measurements of receptor phosphorylation at the single protein level. Several straightforward but important modifications to the SiMPull protocol were developed that improved the reliability of the measurement for phospho-tyrosine detection, including reduction of autofluorescence with NaBH₄ treatment and postfixing of the sample to prevent antibody dissociation. Using the green channel mask to identify receptor locations for calculation of coloc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tubes	MTC Bio	C2000
10 mM Tris-HCl pH 7.4
10 mM Tris-HCl pH 8.0/ 50 mM NaCl			T50 Buffer
100 mm Tissue Culture dish	CELLTREAT	229620	Storage of piranha etched glass/arrays
15 mL conical tube
16% Paraformaldehyde Aqueous Solution	Electron Microscopy Sciences	15710	Hazardous
50 mL conical tube			Functionalized Glass storage/ KOH reuse
50 mM Tris-HCl pH 7.2/150 mM NaCl			Lysis Buffer Component
60 mm Tissue Culture dish	Corning	430166
8% Glutaraldehyde Aqueous Solution	Electron Microscopy Sciences	16020	Hazardous
Acetone (C3H6O)	Millipore Sigma	270725	Hazardous
Alexa Fluor 647 NHS Ester	Thermo Fisher Scientific	A-20006
Animal-Free Recombinant Human EGF	Peprotech	AF-100-15
Anti-Human EGFR (External Domain) – Biotin	Leinco Technologies, Inc	E101
Anti-p-Tyr Antibody (PY99) Alexa Fluor 647	Santa Cruz Biotechnology	sc-7020 AF647
Bath-sonicator	Branson	1200
BCA Protein Assay Kit	Pierce	23227
Biotin-PEG	Laysan Bio	Biotin-PEG-SVA, MW 5,000
Bovine serum albumin	Gold Biotechnology	A-420-1	Tyrode's Buffer Component
Buchner funnel
Bunsen burner
Calcium Chloride (CaCl2)	Millipore Sigma	C4901	Tyrode's Buffer Component
Cell Scraper	Bioworld	30900017-1
Conical Filtering Flask	Fisher Scientific	S15464
Coplin Jar	WHEATON	900470
Countess II Automated Cell Counter	Thermo Fisher Scientific	AMQAX1000
Coverslips 24 x 60 #1.5	Electron Microscopy Sciences	63793
DipImage			https://diplib.org/
DMEM	Caisson Labs	DML19-500
emCCD camera	Andor iXon
Fetal Bovine Serum, Optima	Bio-Techne	S12450H	Heat Inactivated
Fusion 360 software	Autodesk
Geneticin G418 Disulfate	Caisson Labs	G030-5GM
Glacial Acetic Acid (CH3COOH)	JT Baker	JTB-9526-01	Hazardous
Glass serological pipettes
Glass Stir Rod
Glucose (D-(+)-Glucose)	Millipore Sigma	D9434	Tyrode's Buffer Component
Halt Phosphotase and Protease Inhibitor Cocktail (100X)	Thermo Fisher Scientific	78446	Lysis Buffer Component
HEPES	Millipore Sigma	H3375	Tyrode's Buffer Component
Hydrochloric Acid (HCl)	VWR	BDH7204-1	Hazardous
Hydrogen Peroxide (H2O2) (3%)		HX0645
Hydrogen Peroxide (H2O2) (30%)	EMD Millipore	HX0635-2
Ice
IGEPAL CA-630 (NP-40)	Sigma Aldrich	I8896	Lysis Buffer Component
ImmEdge Hydrophobic Barrier Pen	Vector Laboratories	H-4000
Immersol 518F immersion oil	Zeiss	444960-0000-000
in-house vacuum line
L-glutamine	Thermo Fisher Scientific	25030-164
Magnessium Chloride Hexahydrate (MgCl2-6H2O)	MPBio	2191421	Tyrode's Buffer Component
Matlab	Mathworks		Curve Fitting Toolbox, Parallel Computing Toolbox, and Statistics and Machine Learning toolbox
Methanol (CH3OH)	IBIS Scientific	MX0486-1	Hazardous
Milli-Q water
Mix-n-Stain CF Dye Antibody Labeling Kits	Biotium	92245	Suggested conjugation kit
mPEG	Laysan Bio	mPEG-succinimidyl valerate, MW 5,000
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane	UCT United Chemical	A0700	Hazardous
Nanogrid	Miraloma Tech
NeutrAvidin Biotin Binding Protein	Thermo Fisher Scientific	31000
Nitrogen (compressed gas)
NVIDIA GPU with CUDA			Look for compatibility at https://www.mathworks.com/help/parallel-computing/gpu-support-by-release.html
Olympus iX71 Microscope	Olympus
Parafilm M Sealing Film	The Lab Depot	HS234526C
PBS pH 7.4	Caisson Labs	PBL06
PC-200 Analog Hot Plate	Corning	6795-200
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140-163
Phospho-EGF Receptor (Tyr1068) (1H12) Mouse mAb	Cell Signaling Technology	2236BF
Potassium Chloride (KCl)	Millipore Sigma	529552	Tyrode's Buffer Component
Potassium Hydroxide (KOH)	Millipore Sigma	1050330500	Hazardous
Premium PLA Filament, 1.75 mm diameter	Raise 3D	PMS:2035U/RAL:3028	Printing temperature range: 205-235 °C
Pro2 3D printer	Raise 3D
Pyrex 1 L beaker
PYREX 100 mL storage bottles	Corning	1395-100	CH3OH/C3H6O reuse
Pyrex 250 mL beakers
Pyrex 4 L beaker
Quad-view Image Splitter	Photometrics	Model QV2
Refrigerated centrifuge	Eppendorf	EP-5415R
RevCount Cell Counters, EVE Cell Counting Slides	VWR	10027-446
Semrock emission filters: blue (445/45 nm), green (525/45 nm), red (600/37 nm), far-red (685/40 nm)	Semrock	LF405/488/561/635-4X4M-B-000
Serological pipette controller
Serological Pipettes
smite single molecule analysis package			https://github.com/LidkeLab/smite.git
Sodium Bicarbonate (NaHCO3)	Sigma Aldrich	S6014	Hazardous
Sodium Borohydride (NaBH4)	Millipore Sigma	452874	Tyrode's Buffer Component
Sodium Chloride (NaCl)	Millipore Sigma	S9625	Activate by successive heat and pH cycling
Sodium Hydroxide	VWR	BDH3247-1
Sodium Orthovanadate (Na3VO4)	Millipore Sigma	S6508	Hazardous
Sulfuric Acid (H2SO4)	Millipore Sigma	258105	Hazardous
TetraSpeck Microspheres	Thermo Fisher Scientific	T7279	multi-fluorescent beads
Tris (Trizma) base	Millipore Sigma	T1503
Trypan blue stain, 0.4%	Thermo Fisher Scientific	15250061
Trypsin-EDTA 0.05%	Thermo Fisher Scientific	25300120