Waters RapiGest SF Surfactant User Manual

[ Care and Use ManUal ]

RAPIgeSt Sf Surfactant

I. IntroductIon

RapiGest™ SF is a reagent used to enhance in-gel (Ref. 1) and in-solu-

tion (Refs. 2-6) enzymatic digestions of proteins. Recent investigations

also support its use to solubilize glycoproteins prior to deglycosylation

with enzymes such as PNGase F (Publication in preparation). RapiGest

SF helps solubilize proteins making them more susceptible to enzy-

matic cleavage without significantly inhibiting enzyme activity. Unlike

other commonly used denaturants (e.g., SDS or Urea), RapiGest SF

does not modify peptides or suppress endoprotease activity. RapiGest

SF is compatible with enzymes such as Trypsin, Lys-C, Asp-N and Glu-

C and other enzymes. This reagent is easily removed after use allowing

MALDI-TOF MS, HPLC/MS, or HPLC/UV analyses of digested samples.

II. Storage and StabIlIty

The lyophilized powder is stable at room temperature until the expiration

date printed on the label (i.e., 3 years after packaging). Once reconsti-

tuted in high purity water or a buffer (pH 7–10) the solution is stable

for one week when stored at 2–8 °C. Long term storage of frozen ali-

quots is possible but not recommended due to potential solubilization

issues of RapiGest SF or storage buffer.

contentS

I. IntroductIon

II. Storage and StabIlIty

III. reconStItutIon of RAPIgeSt Sf powder

IV. SuggeSted procedure for In-SolutIon dIgeStIonS

V. SuggeSted procedure for In-gel dIgeStIonS

VI. SuggeStIonS for worKIng wItH proteolytIc reSIStant

or HydropHobIc proteInS

VII. SuggeSted SaMple preparatIon for Hplc and MaSS

SpectroMetry

Note: RapiGest SF hydrolyzes in acidic solutions (half life 8 min. at pH

2 and 60 min. at pH 3).

III. reconStItutIon of rapIgeSt Sf powder

Shown in Table 1, are the volumes required to reconstitute the 1 mg

RapiGest SF powder in water or buffer to obtain preferred concentra-

tions. If Mass Spec analysis is required, the recommended buffer is

50mM Ammonium Bicarbonate (NH4HCO3). Alternative buffers, such

as 10 mM Tris-HCl or 25 mM sodium phosphate, are also RapiGest SF

compatible.

RapiGest SF Surfactant 1

[ Care and Use ManUal ]

Table 1: Reconstitution of RapiGest SF Powder

Volume of Buffer Added

to RapiGest SF Vial

1 ml 0.1%

500 µL 0.2%

200 µL 0.5%

100 µL 1%

50 µL 2%

The recommended concentration is 0.1% (w/v) RapiGest SF. Hydro-

phobic proteins may require higher RapiGest SF concentrations (see

note for recommendations for working with proteolytic resistant or

hydrophobic proteins).

RapiGest SF

Concentration (w/v)

IV. SuggeSted procedure for In-SolutIon dIgeStIonS

(Note: Modifications to this suggested method, for example increased

digestion times, may be necessary depending upon the target

proteins.)

1. Suspend the 1 mg of lyophilized RapiGest SF powder in 1 mL of

50 mM Ammonium Bicarbonate (NH4HCO3) to give 0.1% (w/v).

2. Suspend protein pellet in the 0.1% RapiGest SF solution and

vortex.

3. Add DTT to the protein sample to a final concentration of 5 mM.

2. Slice gel spots into <1 mm3 cubes, transfer the gel pieces into a

1.5 ml microcentrifuge tube, add 30 μl of H2O and leave at room

temperature for 15 min.

3. Remove the supernatant, add 20 μl of 50% acetonitrile, mix, and

leave for 15 min.

4. Remove the supernatant, add 30 μl of 100% acetonitrile, mix,

and leave for 15 min.

5. Remove the supernatant, add 20 μl of 0.1 M NH4HCO3, mix, and

leave for 5 min.

6. Add 30 μl of 100% acetonitrile, mix, and leave for 15 min.

7. Remove the supernatant and completely dry the gel pieces with a

Speed Vac.

8. Add 50 μl of 10 mM DTT in 0.1 M NH4HCO3 and incubate at

56 °C for 45 min.

9. Remove the supernatant, add 30 μl of 55 mM iodoacetamide in

0.1 M NH4HCO3 and leave in the dark for 30 min.

(Repeat steps 3–7)

10. Add 20 μl of 0.1% RapiGest SF solution in 50 mM NH4HCO3 and

incubate at 37 °C for 10 min.

11. Remove excess solution and completely dry the gel pieces with a

Speed Vac.

4. Heat the sample at 60 °C for 30 minutes.

5. Cool the sample to room temperature.

6. Add Iodoacetamide to the sample to a final concentration of

15 mM and place the sample in the dark for 30 minutes.

7. Add enzyme for digestion (1:100 to 1:20, w/w).

8. Incubate the samples at 37 °C for (1 hr to overnight depending

upon protein hydrophobicity) for optimum enzymatic digestion.

V. SuggeSted procedure for In-gel dIgeStIonS

(Note: Modifications to this suggested method may be necessary

depending upon the target proteins and applications.)

1. Excise protein spots from gel, wash with 30 μl of H2O and leave

for 15 min.

RapiGest SF Surfactant 2

12. Gradually (single drop at a time) add trypsin (12.5 ng per μL in

50 mM NH4HCO3) to the gel slice until the gel is reswollen.

Continue to incubate on ice for 45 min.

13. Remove excess solution, add 20 μl of 50 mM NH4HCO3 and

incubate at 37 °C overnight.

VI. SuggeStIonS for worKIng wItH proteolytIc reSIStant

or HydropHobIc proteInS

When dealing with hydrophobic proteins such as membrane proteins,

the following modifications are recommended: (See Ref. 3 for more

information)

• Boil the protein/ RapiGest SF mixture at ~ 100 °C for 5 minutes,

cool the sample down before adding proteolytic enzymes.

• Proteins that are highly resistant to protease may require longer

digestion times or higher RapiGest SF concentrations.