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EMARS (Enzyme-Mediated activation of Radical Sources) Methods for Molecular Interaction Analysis
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EMARS (Enzyme-Mediated activation of Radical Sources) Methods for Molecular Interaction Analysis

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Category
Matrices & cellular trafficking
Protocol Name

EMARS (Enzyme-Mediated activation of Radical Sources) Methods for Molecular Interaction Analysis

Authors
Kotani, Norihiro *
Department of Biochemistry, Saitama Medical University

Honke, Koichi
Department of Biochemistry, Kochi University Medical School
*To whom correspondence should be addressed.
KeyWords
Reagents

A compound that specifically binds to an objective cell surface molecule (antibody, lectin etc.)

Filter centrifuge unit (Pall Corporation, Port Washington, NY)

HRP-labeling reagent (kit) (Dojindo Laboratories, Kumamoto, Japan: -NH2 or –SH labeling kit)

PBS

Arylazide-fluorescein (Tokyo Future Style, Inc., Ibaraki, Japan: Ar-Flu)

Tris-HCl (pH 7.4)

Plastic Syringe with 21 to 26 G needle

NP-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 10%

glycerol, and protease inhibitor cocktail (optional))

BCA protein assay kit (Pierce, Rockford, IL)

Anti-fluorescein antibody-conjugated Sepharose (NHS-activated Sepharose (GE Healthcare, Little Chalfont, UK) reacted with goat anti-fluorescein antibody (Rockland Immunochemicals, Gilbertsville, PA; 600-101-096))

Elution reagent (mix with 1%SDS-100mM Tris HCl (pH 7.4) solution and “B

Solution” of MPEX PTS reagent kit (GL Science Inc., Tokyo, Japan) (1:1))

DTT (Wako Pure Chemical Industries, Ltd., Osaka, Japan)

Iode acetoamide (IAA; Wako Pure Chemical Industries, Ltd.)

SDS-eliminant solution (ATTO Corporation, Tokyo, Japan)

Trypsin solution (Trypsin Gold MS spec. grade (Promega Corp., Fitchburg, WI))

C18 cartridge for peptide desalting (e.g. StageTip, ZipTip)

Instruments

Dounce homogenizer

Evaporator (e.g. SpeedVac)

MS system

Methods
1.

Preparation of an EMARS probe

1) 

 Prepare a compound that specifically binds to an objective cell surface molecule (cognitive compound).

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2) 

 Desalt using filter centrifuge unit.

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3) 

 Label the cognitive compound with horseradish peroxidase (HRP) using HRP-labeling reagent (kit).

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4) 

 Stock at 4°C (Freezing condition may weaken HRP activity).

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2.

EMARS method

1) 

 Prepare cultured cells (suspended cells in a microtube or attached cells in a culture dish).

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2) 

 Wash the cells at least twice with PBS.

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3) 

 Add an HRP-conjugated EMARS probe (prepared above) dissolved in appropriate buffer (usually in PBS).

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4) 

 Incubate at a suitable temperature (4°C, 25°C, or 37°C) for 15 min.

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5) 

 Wash the cells three times with PBS.

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6) 

 Add arylazide-fluorescein in PBS (the preferable concentration is about 0.1 mM) and incubate at a suitable temperature (4°C, 25°C, or 37°C) for 10 min.

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7) 

 Lyze the cells by using a disposable plastic syringe equipped with 21 to 26 G needle (10–15 strokes) or Dounce homogenizer.

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8) 

 In the case of attached cells, add 300 μL 100 mM Tris-HCl (pH 7.5) with protease inhibitor cocktail (optional) and then harvest the cells by using a scraper. Transfer the harvested cells to a microtube. In the case of suspended cells, add 300 μLof 100 mM Tris-HCl (pH 7.5) with protease inhibitor cocktail (optional).

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9) 

 Lyze the cells by using a disposable plastic syringe equipped with 21 to 26 G needle (10–15 strokes) or Dounce homogenizer.

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10) 

 Centrifuge at 800 × g for 5 min at 4°C.

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11) 

 Transfer the sup to a new microtube and then centrifuge at 20,000 × g for 15 min at 4°C.

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12) 

 Discard the sup and wash the pellet (microsome fraction) once with 500 μL of 100 mM Tris-HCl (pH 7.5).

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13) 

 Solubilize the pellet with the NP-40 lysis buffer or add chloroform/methanol solution for MS analysis (see Procedure 3).

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14) 

 Measure the total protein concentration in each sample by using BCA protein assay kit.

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15) 

 Apply the samples to several subsequent experiments (SDS-PAGE, immunoprecipitation, antibody array, etc.)

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3.

Analysis of fluorescein-labeled molecules by MS

1) 

 Add 500 μL of chloroform/methanol (2:1 v/v) to the microsome pellet (see 2-12), mix gently, and then add 500 μL distilled water.

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2) 

 Mix gently and centrifuge at 10,000 × g for 5 min at 4°C.

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3) 

 Wash the pellet (you can see in the intermediate layer) with 50% ethanol solution twice.

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4) 

 Remove 50% ethanol solution completely.

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5) 

 Add 100 μL of 1% SDS-100 mM Tris HCl (pH 7.4) and heat at 100°C for 10 min.

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6) 

 Add 400 μL of the NP-40 lysis buffer and mix.

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7) 

 Centrifuge at 20,000 × g for 5 min at 4°C and transfer the sup to a new tube.

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8) 

 Add anti-fluorescein antibody-conjugated Sepharose and rotate tube at 4°C for overnight.

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9) 

 Centrifuge at 800 × g for 5 min at 4°C.

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10) 

 Wash the Sepharose resin 5 times with the NP-40 lysis buffer, twice with 0.5M NaCl in PBS, and then with 50 mM NH4HCO3.

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11) 

 Add 40 μL of the elution reagent to the Sepharose resin and mix.

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12) 

 Heat at 100°C for 5 min.

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13) 

 Transfer the sup to a new tube and add 20 μL of 10 mM DTT/ 50 mM NH4HCO3 solution.

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14) 

 Incubate at 50°C for 30 min.

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15) 

 Add 20 μL of 100 mM IAA/50 mM NH4HCO3 and incubate at 37°C for 1 hr in dark.

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16) 

 Add 5 μL of the SDS eliminant solution (ATTO) and incubate on ice for 30 min.

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17) 

 Centrifuge at 13,000 × g for 10 min at 4°C.

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18) 

 Transfer the sup to a new tube and add 270 µL of 50 mM NH4HCO3

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19) 

 Add 2 μL of 20 mM CaCl2 and 1 μL of the trypsin solution.

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20) 

 Incubate at 37°C for overnight.

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21) 

 Add 350 μL of ethyl acetate and 3.5 μL of trifluoroacetic acid (TFA) and mix gently.

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22) 

 Centrifuge at 20,000 × g for 2 min at room temperature.

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23) 

 Remove the upper layer completely.

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24) 

 Evaporate the sample for 2 h to remove ethyl acetate completely.

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25) 

 Add 350 μL of 5% ACN/0.1%TFA and mix gently.

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26) 

 Desalt the sample using a C18 cartridge for MS analysis (e.g. StageTip).

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27) 

 Apply the sample to MS (LC-MS, MALDI-TOF MS etc.).

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Notes

The information about non-specific labeling of EMARS reaction  

Most important issue about EMARS reaction is now the circumvention of non-specific labeling. The non-specific labeling sometimes make it more difficult to analyze the result. Especially, strong non-specific labeling has been observed in some types of cell lines, such as neural cell and B cell lymphoma cell using arylazide-biotin. Arylazide- fluorescein does not show heavy non-specific reaction, therefore, we now recommend the arylazide-fluorescein. Our study indicated the mechanisms of such non-specific labeling. The arylazide compound penetrates into the cell because of strong hydrophobicity, and then reacts with endogenous peroxidase, maybe existed in the mitochondria and peroxisome, resulting in non-specific labeling of intracellular protein. Our convenient treatment after EMARS reaction may be not enough to remove such intracellular protein. We are investigating the solutions to prevent non-specific labeling using several approaches, however, it is now in progress. Therefore, we would appreciate it if the researchers who use the EMARS reaction pay attention as follows;

1) The most effective way to reduce the background is the separation of plasma membrane proteins from other components. You should adjust the condition of sample preparation after EMARS reaction (lysis method, centrifugation speed etc.) depending on cell lines. Also, you should perform the operations quickly after incubation with arylazide compounds.

 

2) We noticed that the incubation temperature with arylazide is sometimes important for prevention of non-specific labeling in some types of cell lines. Please select proper temperature for your cells and experiments.

Figure & Legends

Figure & Legends

 

 

Fig. 1. Outline of the EMARS-based molecular interaction analysis

The fluorescein-tagged EMARS products are purified and concentrated by immunoaffinity chromatography with anti-fluorescein antibody-immobilized beads and identified by MS-based proteomics analysis.

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