Protocol

In Vivo Isotopic Labeling of Proteins for Quantitative Proteomics

This protocol was adapted from "The Use of Mass Spectrometry in Proteomics," Chapter 8, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Stable isotope coding strategies are of immense value in determining protein concentration changes in cells and tissues triggered by regulatory stimuli (e.g., drugs and toxins) and disease (e.g., caused by mutational changes). Recognizing and identifying the small number of proteins whose expression levels differ as a consequence of disease or external stimuli are complicated by the complexity of biological extracts and the fact that protein post-translational modifications are numerous and can occur at many sites on a protein. One way of quantifying global protein expression patterns involves in vivo labeling. Using the following protocol, stable isotopes can be incorporated into metabolic products, and the relative difference between these products from cells grown in normal or isotope-enriched media can be readily quantified by MS analysis.

MATERIALS

Reagents

Acetone, ice-cold

Acetonitrile (for in-gel protein digestion; see Steps 23-32)

Acetonitrile/H₂O/TFA (66/33/0.1) (for in-gel protein digestion; see Steps 23-32)

Acetonitrile/H₂O/TFA (5/95/0.1)

Ammonium bicarbonate (50 mM) (for in-gel protein digestion; see Steps 23-32)

Ammonium bicarbonate (25 mM) containing 0.1% n-octylglucoside (for in-gel protein digestion; see Steps 23-32)

Ammonium bicarbonate/n-octylglucoside/trypsin solution (for in-gel protein digestion; see Steps 23-32)

Cell culture growth media

The experiment requires growth medium capable of supporting the cells under study (bacterial, yeast, other fungi, insect, or mammalian). The medium must be available in two varieties that are identical except for the inclusion of ¹⁵N-labeled metabolites in one. Two sources of such media are Spectra Stable Isotopes (http://www.spectrastableisotopes.com), which produces the Celtone and Yeastone products; and Cambridge Isotope Laboratories, Inc. (http://www.isotope.com/cil/index.html), which produces the Bio-Express line of growth media.

Growth conditions must be optimized empirically for each cell type and medium. Experience with Bio-Express 1000 medium indicates that it is preferable to add tryptophan to the medium to a final concentration of 40 mg/ml. Bio-Express 1000 media are provided as sterile, 10X concentrates in H₂O. Dilution experiments indicate a linear growth response over the range of 0.25X to 2X concentrations. Because growth rates of Saccharomyces cerevisiae are slightly slower in Bio-Express 1000 medium, prepare a 2X solution as the working stock.

Liquid nitrogen (optional; see Step 14)

ß-Mercaptoethanol (10 mM)

Methanol/acetic acid solution (for in-gel protein digestion; see Steps 23-32)

Potassium ferricyanide/sodium thiosulfate solution, freshly prepared (for in-gel protein digestion; see Steps 23-32)

Protein extraction reagent (e.g., Y-PER reagent, Pierce)

Protease inhibitor cocktail

Reagents for protein separation using 2D gel electrophoresis (see Steps 22.i-22.vii)

For additional details on running 2D gel electrophoresis, see Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: SDS-PAGE of Proteins, Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Rehydration of IPG Strips for Isoelectric Focusing of Proteins, Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Isoelectric Focusing of Proteins in a Multipurpose Flatbed Electrophoresis Unit , Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IEF of Proteins in an IEF-Dedicated Electrophoresis Unit, Preparation of Vertical SDS Slab Gels: Casting a Single Homogeneous Gel, Preparation of Vertical SDS Slab Gels: Simultaneous Casting of Multiple Gradient Gels, and Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IPG Strip Equilibration

Immobiline dry strip gel (Amersham Biosciences)

Polyacrylamide gel (12.5% T, 2.7% C)

Rehydration buffer A

Silver stain reagents for Mass Spectrometry-Compatible Silver Staining

Reagents for protein separation using 2D-HPLC (see Steps 22.viii-22.xvii)

For additional details on running HPLC, see Standard Chromatographic Conditions for RP-HPLC of Proteins.

Ammonium bicarbonate (NH₄HCO₃; 20 mM) containing 5 mM n-octylglucoside

2D-HPLC solvent A

2D-HPLC solvent B

Rehydration buffer B

Mobile phase A

Mobile phase B

Trypsin (0.05-1 µg/µl; see Step 22.xvi)

Reagents for protein separation using HPLC and SDS-PAGE (see Steps 22.xviii-22.xxvi)

For additional details on running ion-exchange HPLC and SDS-PAGE, see Standard Chromatographic Conditions for RP-HPLC of Proteins and SDS-PAGE of Proteins, respectively.

2X gel-loading buffer for HPLC/SDS-PAGE

Mobile phase A

Mobile phase B

Rehydration buffer B

Silver stain reagents for Mass Spectrometry-Compatible Silver Staining

Trichloroacetic acid (TCA) (10%)

Triglycine 8%-16% gradient gel (Invitrogen-NOVEX)

Saccharomyces cerevisiae, freshly grown colonies on a Petri dish

Other types of cells can be labeled using this protocol, provided the growth conditions are adjusted to optimize growth of the cells under study.

Equipment

Centrifugal evaporator (SpeedVac, Thermo Savant, or equivalent)

Centrifuge and rotor (chilled; see Step 11)

Centrifuge tubes (100-ml capacity, chilled; see Step 10)

Dry ice (optional; see Step 14)

Electrophoresis apparatus (for protein separation using HPLC/SDS-PAGE; see Step 22)

Electrophoresis apparatus (Hoefer DALT system, Amersham Biosciences, or equivalent) (for protein separation using 2D gel electrophoresis; see Step 22)

Hemocytometer (optional; see Step 8)

HPLC system (for protein separation using HPLC/SDS-PAGE or 2D-HPLC; see Step 22)

Ice

Incubator with shaker, preset to 30ºC

Incubator, preset to 37°C (for protein separation using 2D-HPLC; see Step 22)

MALDI-TOF mass spectrometer and an LC-MS/MS system

See Step 34, Preparation and Use of an Integrated Microcapillary HPLC Column and ESI Device for Proteomic Analysis, Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS, and Analysis of Complex Protein Mixtures Using Multidimensional Protein Identification Technology (MuDPIT).

Microcentrifuge

Microcentrifuge tubes

Mono-Q column (HR 5/5, Amersham Bioscience, or equivalent) (for protein separation using HPLC/SDS-PAGE or 2D-HPLC; see Step 22)

RP-HPLC column (YMC-Pack CN-AP 4.6 x 150 mm, Ap-502 [YMC, Inc.] or equivalent) (for protein separation using 2D-HPLC; see Step 22)

Sonicator

Spectrophotometer

Tubes (suitable for collecting 2-ml fractions) (for protein separation using HPLC/SDS-PAGE or 2D-HPLC; see Step 22)

Vortex mixer

METHOD

Culturing of Cells

1. Beginning with a freshly grown plate, choose a single yeast colony and inoculate 2 ml of ¹⁵N-labeled culture medium with the colony. Choose a second colony as a control sample, and inoculate 2 ml of unlabeled culture medium with that colony.

2. Vigorously vortex the tubes of medium for ~1 minute to disperse the cells thoroughly.

3. Incubate the two cultures with shaking at 230-270 rpm overnight at 30ºC.

4. Following overnight growth, measure the culture growth with a spectrophotometer. The cultures should be dense (OD₆₀₀ >1.5).

5. Vortex the overnight cultures for ~1 minute to disperse any cell clumps.

6. Inoculate 50 ml of ¹⁵N-labeled medium with the entire overnight culture grown in ¹⁵N-labeled medium. Likewise, inoculate 50 ml of the unlabeled medium with the entire control culture.

7. Incubate with shaking at 220-250 rpm at 30ºC until the cultures have reached mid-log phase (OD₆₀₀ <1.0).

Preparation of Cultures for Protein Extraction

8. Determine the cell density by measuring the culture at 600 nm in a spectrophotometer or by counting cells in a hemocytometer.

9. Using as much of each culture as possible, combine equal numbers of cells from the ¹⁵N-labeled culture and from the unlabeled control culture.

10. Quickly chill the mixture by pouring it into two chilled 100-ml centrifuge tubes filled halfway with ice.

11. Immediately place the tubes in a chilled rotor, and centrifuge them at 1000g for 5 minutes at 4ºC.

12. Pour off the supernatant and resuspend the cell pellet in 50 ml of ice-cold H₂O.
Any unmelted ice pours off with the supernatant.

13. Recover the pellet by centrifugation at 1000g for 5 minutes at 4ºC.

14. Again, discard the supernatant, and immediately freeze the cell pellet by placing the tubes on dry ice or in liquid nitrogen. Store the cells at –80ºC until they are needed.

Preparation of Protein Extracts

15. Add an appropriate volume of protein extraction reagent containing a protease inhibitor cocktail and 10 mM ß-mercaptoethanol to the frozen cell pellet.
Usually 2.5-5 ml of the Y-PER reagent is used for a 1-ml (1 g) cell pellet.

16. Incubate the cells for 30 minutes at room temperature.

17. Spin down the debris and collect the supernatant.

18. Add 5 volumes of ice-cold acetone to the supernatant and vortex the tube.

19. Incubate the protein extract for 10 minutes at –80ºC.

20. Centrifuge the proteins in a microcentrifuge at 20,000g for 5 minutes at 4ºC.

21. Discard the supernatant and immediately carry out Step 22 or store the precipitated proteins at –80ºC until they are needed.

Separation of Proteins

22. Three different ways to separate the protein mixture into individual proteins are 2D gel electrophoresis (Steps 22.i-22.vii), 2D-HPLC (Steps 22.viii-22.xvii), and HPLC combined with SDS-PAGE (Steps 22.xviii-22.xxvi). Perform one of these procedures.
The following is a summary of each respective procedure. For detailed step-by-step instructions, see the links provided in Materials.

2D Gel Electrophoresis

i. Dissolve 500 µg of precipitated protein (from Step 21) in 400 µl of rehydration buffer A.

ii. Rehydrate the proteins on an Immobiline dry strip gel.

iii. Perform isoelectric focusing (IEF) in the first dimension.

iv. Apply the strip to a 12.5% T, 2.7% C polyacrylamide gel and separate the proteins in the second dimension.

v. Following the completion of 2D electrophoresis, stain the gel with silver using a procedure that is compatible with subsequent analysis of the proteins by MS (see Mass Spectrometry-Compatible Silver Staining).

vi. Excise the protein gel spots of interest, and place them in microcentrifuge tubes.

vii. Proceed to Step 23.

2D-HPLC

viii. Dissolve 10 mg of precipitated protein (from Step 21) in 2 ml of rehydration buffer B.

ix. Establish a column flow rate of 1 ml/minute. Set the concentration gradient for mobile phase B at:

• 0-5 minutes, 0% B

• 5-45 minutes, 0%-50% B

• 45-60 minutes, 50%-100% B

x. Collect 30 2-ml fractions in suitable tubes.

xi. Inject each 2-ml fraction into the second HPLC column, a reversed-phase column.
2D-HPLC solvent A is 5% acetonitrile containing 0.1% TFA, and 2D-HPLC solvent B is 95% acetonitrile containing 0.1% TFA.

xii. Establish a column flow-rate of 1 ml/minute. Set the concentration gradient for 2D-HPLC solvent B at:

• 0-5 minutes, 0% B

• 5-60 minutes, 0%-55% B

xiii. Collect 30 2-ml fractions.

xiv. After the second HPLC is complete, dry the fractions in a centrifugal evaporator.

xv. Dissolve the fractions in 20 µl of 20 mM NH₄HCO₃ and 5 mM n-octylglucoside. Vortex and sonicate the samples. Check that the pH is ~8.5.

xvi. Add 1 µl of 0.05-1 µg of trypsin to the fractions. Incubate overnight at 37°C.
The amount of trypsin to use will depend on the protein concentration. The final substrate-to-trypsin ratio should be 50:1.

xvii. Proceed to Step 34.

HPLC/SDS-PAGE

xviii. Dissolve 10 mg of precipitated protein (from Step 21) in 2 ml of rehydration buffer B.

xix. Establish a column flow rate of 1 ml/minute. Set the concentration gradient for mobile phase B at:

• 0-5 minutes, 0% B

• 5-45 minutes, 0%-50% B

• 45-60 minutes, 50%-100% B

xx. Collect 30 2-ml fractions in suitable tubes.

xxi. After the HPLC is complete, precipitate the proteins from each 2-ml fraction with 1 ml of 10% (w/v) TCA. Vortex the tubes and incubate for 30 minutes on ice.

xxii. Centrifuge the tubes at 3000g for 30 minutes at room temperature.

xxiii. Discard the supernatant. Add 2X gel-loading buffer for HPLC/SDS-PAGE, and run each fraction in a separate lane of an 8%-16% gradient triglycine gel.

xxiv. Stain the gels with silver using a procedure that is compatible with subsequent analysis of the proteins by MS (see Mass Spectrometry-Compatible Silver Staining).

xxv. Excise the protein gel spots of interest, and place them in microcentrifuge tubes.

xxvi. Proceed to Step 23.

In-Gel Digestion

23. Destain the silver-stained gel pieces for a few minutes with freshly prepared potassium ferricyanide/sodium thiosulfate solution.

24. Wash the gel pieces vigorously with 500 µl of methanol/acetic acid solution for 30 minutes.

25. Repeat Step 24 four more times, using fresh washing solution each time.

26. Incubate the gel pieces with 500 µl of 50 mM ammonium bicarbonate for 5 minutes.

27. Incubate the gel pieces with 500 µl of acetonitrile for 5 minutes.

28. Dry the gel pieces completely in a centrifugal evaporator.

29. Swell the dried gel pieces in 2 µl of ammonium bicarbonate/n-octylglucoside/trypsin solution.

30. After all solvents have penetrated into the gels (usually 5-10 minutes), add 10 µl of 25 mM ammonium bicarbonate containing 0.1% n-octylglucoside, and allow the mixture to stand for 2 hours at 37ºC.

31. Extract the tryptic peptides twice with 40 µl of acetonitrile/H₂O/TFA (66/33/0.1 [v/v/v]) solution in a 350-W sonicator for 5-10 minutes.

32. Dry the combined extracts (~90 µl) in a centrifugal evaporator.

Mass Spectrometric Analysis of the Proteins

33. Dissolve the dried tryptic peptides in 5 µl of acetonitrile/H₂O/TFA (5/95/0.1 [v/v/v]) solution.

34. Subject 10% of the digest to MALDI-TOF-MS, and use the remainder for LC-MS/MS analysis.
Proteins separated by 2D-HPLC and digested in solution must be passed through a ZipTip prior to MALDI or loaded onto an RP-HPLC column prior to MS/MS.