CEM Discover Proteomics Owner's manual

Brand: CEM

Model: Discover Proteomics

Type: Owner's manual

DISCOVER

PROTEOMICS

INSTALLATION AND OPERATION

INSTRUCTIONS

HARDWARE ASSEMBLY AND SETUP

FIBER-OPTIC TEMPERATURE PROBE1.

Connect the serial cable to the rear of the ﬁso temperature control box and to COM2 in the 1.1.

rear of the Discover (Figure 1).

Connect the blue ﬁber-optic temperature probe into the ﬁso control box, taking care not to 1.2.

bend the probe.

EXTERNAL AIR COOLING2.

On the lower rear side of the Discover install the pressure regulator to the Discover using the 2.1.

¼” polyethylene tubing (Figure 1).

Connect the other side of the pressure regulator to a source of compressed air and set the 2.2.

pressure regulator to 5 PSI.

Figure 1. Instrument Connection Ports.

600790

Rev. 2

6/20/2008

CALIBRATE FIBER-OPTIC PROBE3.

Connect the power cord to the Discover. Locate the power switch (on the left side of the unit) 3.1.

and turn the power ON.

Press EDIT.3.2.

The screen should say “TEMPERATURE”. If yes, press ENTER. Otherwise, use the right 3.3.

arrow key to scroll to “TEMPERATURE” and press ENTER.

Use the right arrow key to scroll to “Select Alternate”.3.4.

Ensure that “DEVICE = FIBER-OPTIC”. If yes, press ENTER. If no, use the right arrow key to 3.5.

change to “DEVICE = FIBER-OPTIC” and press ENTER.

Press EDIT.3.6.

The screen should say “TEMPERATURE”. If yes, press ENTER. Otherwise, use the right 3.7.

arrow key to scroll to “TEMPERATURE” and press ENTER.

Highlight “Enter Calibration” and press ENTER.3.8.

Press EDIT, then enter the 7 digit GF# located on the white band at the base of the ﬁber-optic 3.9.

probe.

Press ENTER. The calibration is now saved in the software.3.10.

Press HOME to return to the Home Screen.3.11.

INSTRUMENT OPERATION

Choose a method.1.

To create a new method:1.1.

Press the Open Folder key.1.1.1.

Use the left arrow key to select “New Method”. Press ENTER.1.1.2.

Press the right arrow key until “Mode = Discover SPS”. Press ENTER.1.1.3.

NOTE: The Discover must be in SPS mode to use the Enzymatic Digestion

attachment. In SPS Mode, the Discover will irradiate the samples with the deﬁned

power only until the maximum temperature is reached, and then cycle the power on

and off to maintain the temperature within the deﬁned variance (delta temperature) for

the remainder of the run.

Set Power, then press ENTER. (50 W is recommended, but may be adjusted. The 1.1.4.

power should be set such that maximum temperature is reached in approximately 4

minutes.)

Set Maximum Temperature, then press ENTER. (55° C is recommended but may be 1.1.5.

adjusted as necessary.)

Set Run Time, then press ENTER. (15:00 (15 min) is recommended but may be 1.1.6.

adjusted.)

Set Delta Temperature to 1° C, then press ENTER.1.1.7.

Set Stirring to “HI”, then press ENTER.1.1.8.

Set Cooling to “ON”, then press ENTER.1.1.9.

Set “Next Stage = (N)”, then press ENTER.1.1.10.

Set “Save Method = (Y)”, then press ENTER.1.1.11.

Create a method name using the arrow keys, highlight “Exit”, and press ENTER. The 1.1.12.

method is now saved in the software.

To use an existing method:1.2.

Press the Open Folder key.1.2.1.

Use right arrow key to scroll to the desired method name, then press ENTER.1.2.2.

Set the system to Open Vessel Mode.2.

Press EDIT.2.1.

Scroll over using the arrow keys until the screen says “Open Vessel” and press ENTER.2.2.

NOTE

The system must be in Open Vessel Mode to use the Discover Proteomics option.

Use the right arrow key to set “Run Open Vessel: Yes” and press Enter.2.3.

Set up the vessel.3.

Add 25mL of either deionized or tap water to the vessel bath bottom. Then add a small 3.1.

magnetic stir bar to the bath (Figure 2).

Lock the vessel holder top in place over the bath bottom.3.2.

Puncture a small hole in the top of a microcentrifuge tube using the needle. This will serve 3.3.

as a temperature control vessel. Fill the tube with the buffer solution used for the digestions.

Place the temperature control vessel into one of the two center holes on the vessel holder.

Insert all desired microcentrifuge sample tubes into the top of the holder. Remove the 3.4.

attenuator and inset the assembled vessel holder into the microwave cavity (Figure 3).

Replace the attenuator and lock in place.

Thread the ﬁber-optic probe through the attenuator opening, and insert into the temperature 3.5.

control vessel. Ensure that the tip of the ﬁber-optic probe is below the solution level in the

temperature control vessel. Lock the attenuator back in place. (Figure 4).

NOTE

Do not ﬁll the temperature control vessel with the bath solution. The microwave heats the bath solution

differently than the buffer solution.

Figure 2. Bath Preparation.

Figure 3. Loading Reaction Vessel into Microwave Cavity.

Figure 4. Temperature Probe.

Press the Play key to run the microwave method as programmed.4.

RECOMMENDED PARAMETERS FOR ENZYMATIC DIGESTION

Power = 50W

Power Mode = SPS

Temperature = 55° C

Delta Temperature = 1° C

Temperature Control = Fiber-optic

Run Time = 15:00

Stirring = Yes—High

Air Cooling = 5 psi

Bath Solution = 25 mL water (deionized or tap may be used)

Enzyme/Protein ratio = 1:100 to 1:10

Temperature Control Sample: Fill the microcentrifuge tube (with hole punctured in top) with the buffer

solution used for the digestions. Place the temperature control vessel

into one of the two center holes on the vessel holder. Do not use the bath

solution—you MUST use the same buffer solution as the samples.

NOTE

It may be necessary to remove the temperature control vessel from the holder to verify that the probe is

below the solution level. The ﬁber-optic probe will not accurately measure the temperature unless the tip

is completely submerged in the solution.

PARTS LIST

SYSTEM

PRODUCT NAME PART #

Discover Proteomics (100-120V/50/60Hz) 925474

Includes the Following:

Bioscience Discover (100-120V/50/60Hz) 908005

Discover Proteomics Accessory Kit 908565

Fiber-Optic Temperature Control 541175

Discover Proteomics (200-240V/50/60Hz) 925476

Includes the Following:

Bioscience Discover (200-240V/50/60Hz) 908010

Discover Proteomics Accessory Kit 908565

Fiber-Optic Temperature Control 541175

ACCESSORIES

PRODUCT NAME PART #

14-place Microcentrifuge Tube Holder 167170

1.5 mL Microcentrifuge Tubes (Qty 50) 169175-M

Micro Stir Bar (pack of 50) 162810

Fiber-Optic Temperature Probe 314325

Regulator Assembly for Air Cooling 541455

Discover Proteomics Operations Manual 600790

Recommended Protocols for Enzymatic Digestion

(adapted from the protocols used by Peter Yau at the University of Illinois)

In-Gel Digestion Protocol

Prepare the following solutions:1.

25 mM Ammonium Bicarbonate (NH1.1. 4HCO3): 100 mg NH4HCO3 per 50 ml water

Reduction Solution: 10 mM dithiothreitol (DTT) in 25 mM NH1.2. 4HCO3 (10 µL 1M DTT to 1 mL 25

mM NH4HCO3, prepared fresh daily)

Alkylation Solution: 55 mM iodoacetamide in 25 mM NH1.3. 4HCO3 (prepare fresh daily)

Destain/Dehydration Solution: 25 mM NH1.4. 4HCO3 in 50% Acetonitrile (ACN)

Extraction Solution: 50% ACN / 5% formic acid1.5.

Crush gel slice with a clean pipet tip or pestel.2.

For Silver stained slices, the gel must be destained (Gharahdaghi et al. 1999). For Coomassie Blue 3.

stained gel slices, proceed to step 4.

Add ~100 4. µL (or enough to cover the gel slice) of the Destain/Dehydration Solution and vortex for 10

min.

Using pipet tip, remove the supernatant and discard. Do not use water aspirator or vacuum pump for 5.

this step!

Repeat steps 4 and 5 once.6.

Speed Vac the gel pieces to complete dryness (~20 min).7.

Add 25 8. µL of the Reduction Solution to each dried gel sample. Incubate at 56° C for 1 hour with shaking

or irradiate with microwaves (50 W) at 55° C for 10 minutes.

Remove supernatant, add 100 9. µL 25 mM NH4HCO3, and rinse once.

Add 50 10. µL of the Alkylation Solution to the sample. Incubate at room temperature in the dark for 1

hour.

Remove supernatant. Rinse sample with 100 11. µL 25 mM NH4HCO3 followed by 2 rinses with 100 µL

Destain/Dehydration Solution. Speed Vac to dryness.

Open a fresh vial of Promega Trypsin (20 12. µg), and add 0.8 mL 25 mM NH4HCO3. Open a fresh vial

of RapiGest™ (Waters), add 0.8 mL 25 mM NH4HCO3. Combine the contents of the RapiGest™ to

Trypsin to give a ﬁnal concentration of 25 ng/µL Trypsin and 0.0625% RapiGest™.

Add 25 13. µL Trypsin/RapiGest™ solution to the sample. Allow the gel pieces to rehydrate for 5-10

minutes. If all the liquid has been absorbed, add an additional 10-25 µL 25 mM NH4HCO3 and allow the

gel pieces to soak for an additional 5-10 minutes. Ideally, there will be a small amount of liquid around

the gel pieces after they have completely rehydrated. Gently vortex and give the sample a quick spin.

Digest in microwave with 50W at 55° C for 15 min.14.

Add 100 15. µL Extraction Solution to sample. Sonicate 30 minutes. Remove supernatant.

NOTE

Before starting, ensure that all surfaces have been cleaned with methanol/water and lint-free cloths,

including the outsides of all tubes (being careful not to remove any labels), the outside and inside of the

Speed Vac and centrifuge, tube racks, bottles, razor blades, etc.

Repeat step 15 two more times, and pool all three extractions. Speed Vac the combined extractions 16.

to dryness.

Dissolve the dried peptides with 10 17. µL 5% ACN / 0.1% formic acid. Sample can be used for MALDI or

ESI analysis.

In-Solution Digest

Prepare the following solutions:1.

25 mM Ammonium Bicarbonate (NH1.1. 4HCO3): 100 mg NH4HCO3 per 50 ml water

Reduction Solution: 10 mM dithiothreitol (DTT) in 25 mM NH1.2. 4HCO3 (10 µL 1M DTT to 1 mL 25

mM NH4HCO3, prepared fresh daily)

Alkylation Solution: 55 mM iodoacetamide in 25 mM NH1.3. 4HCO3 (prepare fresh daily)

Extraction Solution: 50% ACN / 45% H1.4. 2O / 5% formic acid

Add 20 2. µL of the Reduction Solution (prepared fresh) to each sample. Incubate at 56° C for 1 hour with

shaking or irradiate with microwaves (50 W) at 55° C for 10 minutes.

Add 20 3. µL of the Alkylation Solution to the sample. Incubate at room temperature in the dark for 1

hour.

Use Genotech’s Perfect FOCUS™ (product number 786-124) to remove salt, buffer, DTT, and 4.

iodoacetamide. Follow Genotech’s instructions to the precipitation step with Orgasol. After the sample

has been in the freezer for 30 minutes, spin protein sample from Orgasol buffer. Resuspend protein in

25 mM NH4HCO3 to a concentration of around 1-2 µg protein per sample.

Open a fresh vial of Promega Trypsin (20 5. µg), and add 1.6 mL 25 mM NH4HCO3. Add 10-25 µL Trypsin

solution to the samples. The Trypsin to protein ratio should be between 1:10 and 1:100.

Digest in microwave with 50W at 55° C for 15 min.6.

Add 200 7. µL Extraction Solution to sample. Sonicate 30 minutes.

Dissolve the dried peptides with 10 8. µL 5% ACN / 0.1% formic acid. Sample can be used for MALDI or

ESI analysis.

References

Rosenﬁeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992) Anal. Biochem. 203, 173-179.

Hellman, U., Wernstedt, C., Gonez, J., and Heldin, C.-H. (1995) Anal. Biochem. 224, 451-455.

Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., and Mische, S. M. (1999) Electrophoresis

20, 601-605.

NOTE

For digesting in solution, samples should be free from salt and buffer (no more than 25 mM Tris or 50

mM NaCl, etc.). We DO NOT recommend performing the digestion in Tris buffer due to the change in pH

with elevated temperature, which may cause diminished sequence coverage as well as lowered Mascot

scores.

Recommended Reagent Sources

Trypsin: Promega, Roche, or Genotech•

Denaturant: Waters RapiGest™•

Buffer/Salt Removal: Genotech Perfect •

FOCUS™

Tubes: Axygen MAXYMum Recovery™•

Water: Nanopure® or MilliQ®•

CEM Discover Proteomics Owner's manual

CEM Discover Proteomics Owner's manual

CEM Discover Proteomics Owner's manual

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