Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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2.3 Reconstitution of the CyDyes 301

If necessary adjust the pH value with adding

50 mmol/L NaOH solution.

& Note: Proteins have some inherent buffering

capacity and may have decreased the pH value

of the lysis solution below pH 8. Samples which

have been cleaned up with TCA acetone or the

2-D cleanup kit can be quite acidic.

2.3

Reconstitution of the CyDyes

Use 99.8% anhydrous Dimethylformamide (DMF) less than 3

months old from day of opening. The quality of the DMF is critical to

ensure that the protein labeling is successful. The DMF must be

anhydrous and every effort should be used to ensure it is not con-

taminated with water. DMF after opening, over a period of time, will

degrade with amine compounds being produced. Amines will react

with the NHS ester CyDye reducing the concentration of dye available

for protein labeling. Adding a 4  molecular sieve to DMF during sto-

rage is a good measure to prolong the useful lifetime of DMF.

2.3.1

For Minimal Labeling of Lysines

CyDye fluors for minimal labeling are available in different package

sizes. Figure 2.1 shows a schematic overview over dye reconstitution

and labeling for the smallest package size (5 nmol).

Dye stock solution: CyDye DIGE Fluor minimal dyes solid com-

pounds are reconstituted in Dimethylformamide (DMF) giving a con-

centration of 1 nmol/mL (e.g. 25 mL DMF to 25 nmol/L of dye). The

stock solution of Cy2 will have a deep yellow, Cy3 a deep red, and Cy5

a deep blue color.

Take a small volume of DMF from its original

container and dispense into a microcentrifuge

tube.

Take the CyDye from the –20 C freezer and

leave to warm for 5 minutes at room tempera-

ture

After 5 minutes add 25 mL of the DMF to each

new vial of CyDye.

Replace the cap on the dye microcentrifuge tube

and vortex vigorously for 30 seconds.

The quality of the DMF is the

major source of error for inade-

quate labeling.

The solutions are stable at

–20 C for several months.

Step 2: Fluorescence Difference Gel Electrophoresis302

Centrifuge the microcentrifuge tube for 30 sec-

onds at 12,000 g in a benchtop microcentrifuge.

The dye can now be used.

& Note: When dyes are not being used they should

be returned to the –20 C freezer as soon as

possible and stored in the dark.

Fig. 2.1: Schematic overview over CyDye reconstitution

and labeling for 5 nmol packages for minimal labeling.

Dye working solution: Dilute 1 volume of the stock CyDye in 1.5

volumes of high grade DMF.

Briefly spin down dye stock solution in a micro-

centrifuge.

Add 7.5 mL of DMF to a sterile microcentrifuge

tube.

Add 5 mL of the stock dye solution.

Now there are 5,000 pmol CyDye in 12.5 mL; therefore 1 mL contains

400 pmol.

& Note: CyDye in the diluted form is only stable

for 2 weeks at –20 C.

10 mmol/L lysine solution:

Reagent Quantity Final concentration

L-Lysine (MW 182.6) 18 mg 10 mmol/L

Make up to 10 ml with distilled water

Store at –20 C. Stable for 6 months.

After reconstitution CyDye is

only stable and useable until

the expiry date listed on the

tube.

2.4 Minimal Labeling of Lysines 303

& Note: The pipetting error should be kept to a

minimum. Therefore, depending on the number

of samples to be labeled, the dyes can be directly

reconstituted to a working solution.

2.3.2

For Saturation Labeling of Cysteines

Dye solution:

In principle the procedure is like with minimal dyes,

just with different amounts.

CyDye DIGE Fluor saturation dyes solid com-

pounds are reconstituted in dimethylformamide

giving a concentration of 2 mmol/L (50 mL DMF

to 100 nmol of dye). The stock solution of Cy3

has a deep red and Cy5 a deep blue color. It is

stable at –20 C for two months maximum. Here

the solution is not further diluted for labeling.

2 mmol/L TCEP (Tris carboxyethyl phosphine):

Reagent Quantity Final concentration

TCEP (MW 286.7) 5.7 mg 2 mmol/L

Make up to 10 mL with distilled water

2.4

Minimal Labeling of Lysines

Conditions:

For efficient labeling the cell lysate (protein sam-

ple) should have optimally pH 8.5.

Labeling temperature is 0 C.

The protein concentration should be between

5 mg/mL and 10 mg/mL.

Sample must be free of IPG buffers or carrier

ampholytes and reductants.

Preparing the pooled internal standard: Take the same amount of

aliquot from each sample and mix them together. A minimum of an

equivalent of 50 mg protein internal standard per gel is required.

If mass spectrometry analysis requires a higher protein load than

150 mg in a gel, a higher volume of standard is collected and divided

For the example in Figure 2.1

the dye would be directly recon-

stituted with 12.5 mL DMF.

The stock solution is used as

working solution.

It must be above pH 8.0.

It must be between 1 mg/ml

and 20 mg/ml.

Step 2: Fluorescence Difference Gel Electrophoresis304

into two portions: one is labeled with Cy2; the other portion is left

unlabeled. The required amount of unlabeled standard is later on

spiked into the mixture of labeled samples and standard before appli-

cation on the IPG strip.

Samples and dyes must be mixed well by rigorously pipetting.

Insufficient mixing could lead to preferential labeling of some pro-

teins. The sample must not get frozen, because this would cause pre-

ferential labeling of some proteins. Since cell lysates are viscous, it is

important to mix samples thoroughly in all mixing steps to avoid

non-uniform labeling.

Internal standard labeling with Cy2:

Add a volume of internal standard equivalent to

n50mg protein to a sterile microcentrifuge

tube.

Add n mL of diluted Cy2 to the microcentrifuge

tube containing the pooled standard (i.e. 300 mg

of protein is labeled with 2,400 pmol of dye).

Mix and centrifuge briefly in a microcentrifuge.

Leave on ice for 30 minutes in the dark.

Add n mL of 10 mmol/L lysine to stop the reac-

tion. Mix and spin briefly in a microcentrifuge.

Leave for 10 minutes on ice in the dark.

Labeling is now finished.

Standards can now be stored for at least three

months at –70 C in the dark.

Sample labeling with Cy3 and Cy5:

Add a volume of sample equivalent to 50 mg pro-

tein to a sterile microcentrifuge tube.

Add 1 mL of diluted CyDye to the microcentri-

fuge tube containing the protein sample (i.e.

50 mg of protein is labeled with 400 pmol of dye).

Mix and centrifuge briefly in a microcentrifuge.

Leave on ice for 30 minutes in the dark.

Add 1 mL of 10 mmol/L lysine to stop the reac-

tion. Mix and spin briefly in a microcentrifuge.

Leave for 10 minutes on ice in the dark.

Labeling is now finished.

Samples can now be stored for at least 3 months

at –70 C in the dark.

Vortexing is not recommended.

n is the number of gels in the

experiment.

2.5 Saturation Labeling of Cysteines 305

2.5

Saturation Labeling of Cysteines

The complete procedure takes about 2 hours.

Conditions:

For efficient labeling the cell lysate (protein sam-

ple) must have pH 8.0.

Labeling temperature is 37 C.

The reductant TCEP: dye concentration ratio is

always kept at a 1:2 ratio to ensure efficient label-

ing.

The protein concentration should be 0.55–

10 mg/mL.

For samples containing proteins with high

cysteines content, more TCEP and dye are

needed.

Mostly 5 mg sample proteins are labeled and analyzed as proposed

below. However in the paper by Sitek et al. (2005) labeling of 2.5 mg

protein – corresponding to 1,000 pancreatic tissue cells – with the

recommended amounts of reductant–dye has been described.

Preparing the pooled internal standard: Take the same amount of ali-

quot from each sample and mix them together. For cysteine labeling

mostly an equivalent of 5 mg protein internal standard per gel is used.

For the internal standard as well as for the preparative gel Cy3 is

preferred to Cy5, because Cy3 shows lower self-quenching effects in

the high molecular weight region than Cy5. Therefore the image of

the preparative gel will be more similar to the image of the pooled

standard for optimal spot picking.

Samples and dyes must be mixed well by rigorously pipetting.

Insufficient mixing could lead to preferential labeling of some pro-

teins. The sample must not get frozen, because this would cause pre-

ferential labeling of some proteins. Since cell lysates are viscous, it is

important to mix samples thoroughly by snipping at the tube in all

mixing steps to avoid non-uniform labeling.

Internal standard labeling with Cy3:

Add a volume of internal standard equivalent to

n5mg to a sterile microcentrifuge tube.

Make up to n9mL with lysis solution.

Add n1mL of 2 mmol/L TCEP.

Vortexing is not recommended.

n is the number of gels in the

experiment.

Assuming a cysteine content of

2%.

Step 2: Fluorescence Difference Gel Electrophoresis306

Mix vigorously by pipetting and spin.

Incubate at 37 C for 1 hour in the dark.

Add n2mL of 2 mmol/L Cy3 saturation dye

solution.

Mix vigorously by pipetting and spin.

Incubate at 37 C for 30 minutes in the dark.

Stop the reaction by adding an equal volume of

2 sample solution.

Mix vigorously by pipetting and spin.

Labeling is now finished.

The labeled samples can be stored for 1 month

at –70 C in the dark.

Sample labeling with Cy5:

Add a volume of protein sample equivalent to 5

mg to a microcentrifuge tube.

Make up to 9 mL with lysis solution.

Add 1 mL of 2 mmol/L TCEP.

Mix vigorously by pipetting and spin.

Incubate at 37 C for 1 hour in the dark.

Add 2 mL of 2 mmol/L Cy5 saturation dye solu-

tion.

Mix vigorously by pipetting and spin.

Incubate at 37 C for 30 minutes in the dark.

Stop the reaction by adding an equal volume of

2 sample solution.

Mix vigorously by pipetting and spin.

Labeling is now finished.

The labeled samples can be stored for 1 month

at –70 C in the dark.

Preparative sample labeling with Cy3: The protein amount of the

reference proteome applied on the preparative gel is usually 300 mg.

Add a volume of protein sample equivalent to

300 mg to a microcentrifuge tube.

Add 60 mL of 2 mmol/L TCEP.

Mix vigorously by pipetting and spin.

& Note: Cell lysates are viscous. Therefore it is

important to mix samples thoroughly in this and

all following mixing steps to avoid non-uniform

labeling.

Incubate at 37 C for 1 hour in the dark.

Corresponding to n 4 nmol/L.

Assuming a cysteine content of

2%.

Corresponding to 4 nmol/L.

Assuming a cysteine content of

2%.

2.6 Preparation for Loading the Samples onto IPG Strips 307

Add 120 mL of 2 mmol/L Cy3 saturation dye

solution.

Mix vigorously by pipetting and spin.

Incubate at 37 C for 30 minutes in the dark.

Stop the reaction by adding an equal volume of

2 sample solution.

Mix vigorously by pipetting and spin.

Labeling is now finished.

The labeled samples can be stored for 1 month

at –70 C in the dark.

Saturation labeling optimization: Because it is impossible to predict

the cysteines content of the protein mixture contained in a sample

type, it may be necessary to optimize the amount of reductant–dye

per protein. In a “same/same experiment” aliquots of the pooled sam-

ples are labeled with the same amounts of the different CyDyes, com-

bined, and separated with a 2-D gel. After scanning the images of the

Cy3 and Cy5 channel, the patterns are overlaid with ImageQuant

or PaintShop Pro and inspected in a false color image:

When the spots are matching perfectly, the

amount of reductant–dye is correct for this parti-

cular sample type.

When red spots are visible, which are horizon-

tally offset from some of the green spots, overla-

beling has occurred: there is some unspecific

labeling of lysines which alter the isoelectric

point.

When there are vertical spot trains and/or

streaks, some proteins have been underlabeled:

partially labeled proteins migrate faster in SDS

electrophoresis than more or fully labeled pro-

teins.

The reductant–dye amount needs to be corrected accordingly. When

sufficient pooled sample material is available, a dilution series experi-

ment can be performed, usually in the range from 1 nmol reductant

for 2 nmol dye to 4 nmol/L reductant for 8 nmol dye.

2.6

Preparation for Loading the Samples onto IPG Strips

2 Sample solutions:

The Pharmalytes 3–10 work very well for wide

pH gradients and the pH gradient 4–7. For basic gradients or narrow

When the sample is very

limited, a reference proteome

has to be found for performing

the optimization procedure.

The little size differences are not

resolved.

The pI is not affected.

Step 2: Fluorescence Difference Gel Electrophoresis308

gradients the Pharmalytes 3–10 are replaced by the related IPG buf-

fers.

2 Sample solution 1: 9 mol/L urea, 2% (w/v) CHAPS, 1% (v/v)

Pharmalytes pH 3–10 (or IPG buffer respective to the pH gradient),

2% (w/v) dithiothreitol (DTT), 0.01% (w/v) Bromophenol blue.

2 Sample solution 2: 2 mol/L thiourea, 7 mol/L urea, 2% (w/v)

CHAPS, 1% (v/v) Pharmalytes pH 3-10 (or IPG buffer respective to

the pH gradient), 2% (w/v) dithiothreitol (DTT), 0.01% (w/v) Bromo-

phenol blue.

After the protein samples have been CyDye-

labeled, add an equal amount of 2 sample solu-

tion and leave on ice for 10 minutes.

Combine the labeled samples and pooled inter-

nal standards according to the experimental

design.

Apply them onto the IPG strip as explained in

the next Chapter.

309

Step 3: Isoelectric Focusing

Disposable gloves must be worn during each step to avoid contamina-

tion of the sample.

& Note: It is very important that each sample is

centrifuged before application onto the IPG

strip!

Choose 18 cm IPG strips instead of 24 cm strips, when molecular

weight markers and 1-D samples should be applied onto the SDS gel.

The “worst case” conditions are given here for running the strip.

These “worst case” conditions are applied when a novel sample type

has to be separated, whose behavior in the electric field is unknown.

IPG strips with printed serial numbers are very useful for tracing

the samples through the 2-D electrophoresis procedure.

Generally, create a table with a list of all IPG

strip numbers used, and fill in which sample

will be applied on which IPG strip, as shown in

Table 3.1.

Tab. 3.1: IPG strip numbers and sample trace chart (example).

IPG strip number Sample with

Cy3 label

Sample with

Cy5 label

Internal standard

with Cy2 label

197 514 Control 1 Treated 6 All 24 samples

197 515 Control 2 Treated 5 All 24 samples

197 516 Control 3 Treated 4 All 24 samples

197 517 Control 4 Treated 3 All 24 samples

197 518 Control 5 Treated 2 All 24 samples

197 519 Control 6 Treated 1 All 24 samples

197 520 Treated 7 Control 12 All 24 samples

197 521 Treated 8 Control 11 All 24 samples

197 522 Treated 9 Control 10 All 24 samples

197 523 Treated 10 Control 9 All 24 samples

197 524 Treated 11 Control 8 All 24 samples

197 525 Treated 12 Control 7 All 24 samples

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

We assume that the reader of

this book is starting with 2-D

electrophoresis or looking for

some advice to improve the

quality of the 2-D patterns.

Step 3: Isoelectric Focusing310

3.1

Reswelling Tray

This device is used for both, “passive” rehydration loading of samples

directly into the IPG strips or pre-rehydration of IPG strips for cup

The next three tables list rehydration solutions optimized for differ-

ent sample and pH gradient types.

Tab. 3.2: Standard rehydration solution.

8 mol/L urea 4.8 g

0.5% (w/v) CHAPS 50 mg

10% (v/v) glycerol 1.2 mL

1.25% (v/v) Pharmalytes 3–10 *) 125 mL

0.002% Bromophenol blue **) 10 mL

Water, deionized, fill up to 10 mL

*) or IPG buffer of respective pH interval

**) from 1% Bromophenol blue solution

The rehydration solution can be produced in larger quantities with-

out the DTT and stored frozen in aliquots at a temperature at –20 C

or colder.

The DTT should be added before use: 0.28% (w/v).

& Note: If the sample was extracted or solubilized

in presence of thiourea, the rehydration solution

must also contain thiourea.

Tab. 3.3: Hydrophobic proteins rehydration solution.

7 mol/L urea 4.2 g

2 mol/L thiourea 1.5 g

0.5% (w/v) CHAPS 50 mg

10% (v/v) glycerol 1.2 mL

1.25% (v/v) Pharmalytes 3–10 *) 125 mL

0.002% Bromophenol blue **) 10 mL

Water, deionized, fill up to 10 mL

*) or IPG buffer of respective pH interval

**) from 1% Bromophenol blue solution

The DTT should be added before use: 0.28 % (w/v).

For rehydration loading dilute sample accordingly.

As described in Section 1.5.1, Sample preparation, on pages 56 and

79 for basic gels, the addition of DeStreak instead of a reductant pre-

Passive rehydration loading

means without applying an

electric field.

Repeated freeze thawing must

be avoided.

For acidic pH gradients the

addition of DeStreak has no

value.