Download - Abstract An important goal of the AfCS Protein Chemistry Laboratory is the analysis of ligand-induced changes in protein phosphorylation. Stable Isotope.

Abstract

An important goal of the AfCS Protein Chemistry Laboratory is the analysis of ligand-induced changes in protein phosphorylation. Stable Isotope Labeling with Amino acids in Cell culture (SILAC) is a recently developed method that is likely to be a major advance in quantitative proteomics. This poster describes the establishment of SILAC-related methods for the quantitative analysis of protein phosphorylation. RAW 264.7 cells expressing a variety of FLAG-tagged versions of signaling proteins were grown in media containing normal (light) or 13C(6)-labeled arginine (heavy). Cells grown in light medium were left untreated and cells grown in heavy medium were stimulated with ligands or treated with phosphatase inhibitors. SILAC quantification experiments are conducted by comparing the amount of individual peptides or phosphopeptides present in control samples with the amount present in a sample from stimulated cells. The ratio of light to heavy peptide ion intensities was determined by mass spectrometry. Time course experiments showed that incorporation of [13C]arginine into RAW cell proteins was complete after 4 days of culture in heavy medium. Mass spectrometry methods to accurately quantify light to heavy ratios were optimized and verified in a series of experiments where extracts from cells grown in normal or heavy media were mixed at different ratios. Preliminary work on quantifying ligand-induced changes in phosphorylation has focused on FLAG-Erk1and FLAG-Akt1. Our intitial experiments have focused on optimizing methods for detection of the appropriate phosphopeptides from these proteins.

Introduction

Due to recent developments in the field of quantitative proteomics, the use of stable isotope labeling and mass spectrometry to quantify changes in protein phosphorylation became an attractive technology for the AfCS Protein Chemistry Laboratory. SILAC is a method that is likely to be a major advance in quantitative proteomics. Although the principles of SILAC are not new, the combination of methods used in the procedure provides a relatively straightforward way to determine the relative abundance of peptides by mass spectrometry. SILAC allows quantitative assessment of changes in protein expression, changes in protein covalent modifications (e.g., phosphorylation), and changes in ligand-induced formation of protein complexes. A major application of SILAC within the AfCS will be quantification of ligand-induced changes in phosphorylation of FXM proteins. Toward this end, retroviruses expressing FLAG-tagged versions of Erk1, Grb2, Akt1, SHP2, Grk2, and Syk have been constructed. These viruses were used to infect RAW cells which were subsequently selected for drug resistance to generate stable cell populations expressing the epitope-tagged proteins. Immunoprecipitation with anti-FLAG antibodies showed that the selected cells express relatively large amounts of tagged proteins and should be suitable for the SILAC experiments.

Method

Conclusions

• A protocol for the effective labeling cells with heavy isotopes was established.

• Four days of labeling are needed for complete incorporation of heavy isotopes into RAW proteins.

• Labeling lysines, in addition to arginine, will produce more peptide pairs for the calculation of H/L ratio.

• Variation in heavy to light ratio calculations were decreased by averaging multiple MS scans.

• Detection of the relevant phosphopeptides for SILAC experiments varies with different phosphopeptides.

• Absolute quantification of protein phosphorylation can be achieved using AQUA peptides as an internal standards

Figure 5. Detection of ligand-stimulated ERK1 phosphorylation by mass spectrometry. RAW 264.7 cells stably expressing FLAG-Erk1 were either left untreated or treated with 500 pM LBP and 200 ng/ml LPS for 15 min. Following treatment, the cells were lysed in buffer containing 0.5% NP-40. The lysates were homogenized, ultracentrifuged, and filtered through a 0.45um syringe filter. The lysates were incubated overnight with anti-FLAG M2 agarose. The beads were washed with a high salt buffer, then with lysis buffer, and eluted 3 times with 0.1 mg/ml 3X-FLAG peptide in TBS. The eluted proteins were resolved by SDS-PAGE and the band corresponding to FLAG-Erk1 was excised and digested with trypsin. The tryptic peptides were then analyzed in negative ion mode by nanospray mass spectrometry. Panels A, B, and C show results from control cells; panels D, E, and F show results from LPS/LBP-stimulated cells. Panels A and D show results of precursor ion scanning (to specific detect phosphopeptides). Panels B and E show the peptide ion intensities in the m/z region corresponding to the singly phosphorylated (1p) Erk1 phosphopeptide; panels C and F show peptide ion intensities from the m/z range corresponding to the doubly phosphorylated (2p) Erk1 peptide.

Figure 2. 13C-Arginine Labeling Time Course of RAW 264.7 Cells. The time course of 13C-arginine labeling of RAW cell proteins was monitored by detecting the light and heavy ion pair from a peptide derived from Hsp90 peptide. Cells were grown in normal medium (day 0) or for increasing periods in medium where normal arginine was replaced with [13C]arginine (day 1-day3).

Figure 6. Detection of the Akt1 phosphopeptide from calyculin-A treated RAW cells. RAW cells stably expressing FLAG-Akt1 were stimulated with M-CSF and FLAG-Akt1 was isolated, digested with trypsin and analyzed by mass spectrometry. Panel A: negative ion mode precursor scan detects the Ser473 phosphopeptide. Panel B: full MS scan in negative ion mode detects a peak at m/z 864.72 corresponding to Ser473 peptide. Panel C: full MS scan in positive ion mode detects a small peak corresponding to Ser473 peptide at m/z 867.57. Panel D: MS/MS fragmentation pattern of the parent ion at m/z 867.57 identifies the singly phosphorylated Ser473 peptide.

Figure 7. Quantitative Analysis of the Akt1 Ser473 phosphopeptide using an Internal Standard (Aqua peptide). RAW cells stably expressing FLAG-Akt1 were treated with calyculin-A and FLAG-Akt1 was isolated, digested with trypsin and analyzed by mass spectrometry. The AQUA peptide, which is 10 Da heavier than the native target peptide, was added before digestion, and extracted with the native peptide. The peptides were then analyzed by nanospray mass spectrometry. The ion intensities of the doubly charged native Ser473 phosphopeptide and the AQUA phosphopeptide are shown.

Quantitative Analysis of Protein Phosphorylation in RAW 264.7 Cells Using SILAC and AQUA PeptidesShu H, Bi Q, Cox, R, Draper L, El Mazouni F, Lyons K, Mumby M, Sethuraman D, Brekken D

Alliance for Cellular Signaling Laboratories, University of Texas Southwestern Medical Center, Dallas, TX

+TOF MS: 1.817 to 7.818 min from hs040414_SILAC_Protein_467_Day3_3A.wiffa=3.58510353443061910e-004, t0=3.20920165373026980e+001

Max. 567.3 counts.

755.0 756.0 757.0 758.0 759.0 760.0 761.0 762.0 763.0 764.0 765.0m/z, amu

0

5

10

15

20

25

30

35

40

42

Inte

ns

ity, c

ou

nts

760.6319

761.1297

761.6246

762.1236757.5946 763.5809755.5774 764.5798758.5864756.5751 759.5683 760.1281

763.0774756.0610

day3H


Max. 951.6 counts.

755.0 756.0 757.0 758.0 759.0 760.0 761.0 762.0 763.0 764.0 765.0m/z, amu

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

25.2

Inte

ns

ity, c

ou

nts

760.6201

761.1215

761.6160

757.6033

758.1087 764.0547758.5869755.5809 762.0987765.0367763.5799

756.5613 759.5730 760.0842 763.0434759.0714

H

L

L

day2

+TOF MS: 0.817 to 5.018 min from Sample 2 (Positive MS) of hs040414_SILAC_Protein_467_Day1...a=3.58510353443061910e-004, t0=3.20920165373026980e+001

Max. 514.6 counts.

755.0 756.0 757.0 758.0 759.0 760.0 761.0 762.0 763.0 764.0 765.0m/z, amu

0

2

4

6

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22

24

26

28

30

32

Inte

ns

ity, c

ou

nts

760.6151

757.6045

761.1203758.1092

761.6098758.6022

755.5810764.5780

763.5638759.0533 762.0913 764.0617756.5557 759.5726

755.0444


Max. 270.1 counts.

755.0 756.0 757.0 758.0 759.0 760.0 761.0 762.0 763.0 764.0 765.0m/z, amu

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

Inte

ns

ity, c

ou

nts

757.6151

758.1204

758.6155

755.5783 760.5935 761.5872759.5942 764.5780756.5713 762.5709759.1287 763.5749

755.0874 764.1196756.0558

LL H day1day0

-TOF MS: 0.033 to 0.534 min from Sample 3 (Negative MS) of hs040317_Protein_449_Akt_CLA_Tr...a=3.58623901186383460e-004, t0=6.39034864398807510e+001

Max. 854.6 counts.

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z, amu

0

50

100

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200

250

300

350

400

450

500

550

600

650

700

750

800

850

Inte

ns

ity, c

ou

nts

635.1837

643.1786

681.2843828.3786

899.7992

864.7202

818.8885

964.3430761.2618

890.5584601.1922 819.5530 1228.8401

973.3439588.1837 1271.3968486.1625 895.8882 1058.8050664.2337329.1596 951.2689 1176.3417 1344.3488607.2034

+TOF MS: 0.135 to 1.334 min from Sample 8 (Positive MS) of hs040317_Protein_449_Akt_CLA_T...a=3.58510353443061910e-004, t0=3.20920165373026980e+001

Max. 1112.8 counts.

400 500 600 700 800 900 1000 1100 1200 1300m/z, amu

0

100

200

300

400

500

600

700

800

900

1000

1100

Inte

ns

ity, c

ou

nts

637.4326

630.5546

415.8580601.7700

645.4383

645.9389

416.3605821.2058

590.4307

545.7099 517.4561 830.7107376.2806 707.4813867.5723426.8530 1073.7392615.4291 896.6338 1273.90711231.3157455.8422 804.5474

+TOF Product (866.6): 118 MCA scans from Sample 11 (Positive MSMS 866.61) of hs040317_Pro...a=3.58510353443061910e-004, t0=3.20920165373026980e+001

Max. 1017.0 counts.

100 200 300 400 500 600 700 800 900 1000 1100 1200m/z, amu

0

50

100

150

200

250

300

350

400

450

500

550

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650

700

750

800

850

900

950

1000

Inte

ns

ity, c

ou

nts

254.2307

538.4286

818.0975510.4263373.3084

809.0869763.5776391.3191 606.4451490.3730195.1676173.1449

230.1769 867.0894773.5589694.5090388.2881 1097.7482520.3973246.1771 493.3773237.2022 997.7566726.4972 849.0863147.1672

full MS scan(negative mode)

full MS scan(positive mode)

fragmentation of m/z 867.57(positive mode)

RPHFPQFpSYSASGTA

TOF PreCursor 10.002 min from Sample 5 (Negative Precursor 700-1200) of hs040317_Protein_449... Max. 89.0 counts.

700 750 800 850 900 950 1000 1050 1100 1150 1200m/z, amu

0

5

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45

50

55

60

65

70

75

80

85

89

Inte

ns

ity, c

ou

nts

865.0543

905.2512

731.9238 785.1818 985.91251098.2667 1178.3834710.3400 876.2714867.4667 893.3375 1046.6750

Precursor scan(negative mode)

phosphopeptide at m/z=865.72m/z 864.72 (M)-2

phosphopeptide at m/z=865.05 (M)-2

phosphopeptide atat m/z=867.57 (M+2H)+2

= Ser473 peptide phos at S473+ some phos at S475

Figure 4. Expression, Stimulation and Immuno-affinity Isolation of Flag-tagged FXM Proteins. RAW 264.7 cells were infected with retroviruses expressing epitope-tagged Akt1 or Erk1 and selected for puromycin resistance. Selected cell populations were either left untreated (C) or treated with the following ligands or inhibitors: calyculin-A (CL-A), M-CSF or LBS plus LBP (LPS/LBP). Following the treatment, the cells were lysed in buffer containing 0.5% NP-40. The lysates were homogenized, ultracentrifuged, and filtered through a 0.45um syringe filter. The lysates were incubated overnight with anti-FLAG M2 agarose. The beads were washed with a high salt buffer, then with lysis buffer, and eluted 3 times with 0.1 mg/ml 3X-FLAG peptide in TBS. One aliquot of the anti-FLAG eluates were resolved on a 10% SDS gel and transferred to a membrane and blotted with an anti-phospho-specific antibodies (anti Ser473 for Akt and anti-Thr202/Tyr204 for Erk1). A second larger aliquot was resolved by SDS-PAGE and stained with colloidal Commassie Blue.

-TOF MS: 0.067 to 1.034 min from Sample 3 (Negative MS) of hs040519_ProteinLab_497_Akt1_CL...a=3.58623901186383460e-004, t0=6.39034864398807510e+001

Max. 252.7 counts.

861.0 862.0 863.0 864.0 865.0 866.0 867.0 868.0 869.0 870.0 871.0 872.0 873.0 874.0 875.0m/z, amu

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

25.3

Inte

ns

ity, c

ou

nts

869.8419864.8276

865.3320

870.3496

865.8330

870.8459

866.3366 869.3517

860.4393 871.3758863.3722

Figure 1. Stable Isotope Labeling by Amino acids in Cell culture (SILAC). A summary of the method to detect relative quantitation of phosphorylation from Raghothama and Pandey, Trends Biotechnol. 2003 Nov; 21(11): 467-70 is shown in Panel A. The applications of SILAC to current Protein Lab projects are diagrammed in Panel B.

A

C

B

D

Results

References

• Raghothama C and Pandey A. Trends Biotechnol, 2003, 21(11): 467-70.

• Ong SE, Kratchmarova I, Mann M. Journal of Proteome Research, 2003, 2(2):173-81.

• Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Molecular & Cellular Proteomics, 2002, 1(5):376-86.

+TOF MS: 7.018 to 10.886 min from hs040423_SILAC_Protein_482_2D_L-H_1-2.wiffa=3.58510353443061910e-004, t0=3.20920165373026980e+001

Max. 805.4 counts.

748.0 749.0 750.0 751.0 752.0 753.0 754.0 755.0 756.0 757.0 758.0m/z, amu

0

10

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60

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90

100

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120

130

140

150

160

Intensity, counts

753.5649

754.0681

750.5583

+TOF MS: 19.288 to 25.339 min from hs040423_SILAC_Protein_482_2E_L-H_2-1.wiffa=3.58510353443061910e-004, t0=3.20920165373026980e+001

Max. 288.0 counts.

748.0 749.0 750.0 751.0 752.0 753.0 754.0 755.0 756.0 757.0 758.0m/z, amu

0

20

40

60

80

100

120

140

160

180

200

211

Intensity, counts

750.5759

751.0782

753.5872

751.5788754.0873

754.5905

752.0809

L/H=1/2

+TOF MS: 3.401 to 8.952 min from hs040423_SILAC_Protein_482_2C_L-H_1-1.wiffa=3.58510353443061910e-004, t0=3.20920165373026980e+001

Max. 1615.5 counts.

748.0 749.0 750.0 751.0 752.0 753.0 754.0 755.0 756.0 757.0 758.0m/z, amu

0

5

10

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20

25

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50

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65

70

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80

85

90

95

99

Intensity, counts

750.5531

753.5639

751.0553

754.0669

751.5633

754.5676

L/H=1/1 L/H=2/1

Figure 3. Detection of Light/Heavy Ratios from Mixed Sample. Lysates from RAW cells grown in light or heavy medium were mixed at ratios of 1:1, panel A; 2:1, panel B; or 1:2, panel C). The lysates were resolved by SDS PAGE and the protein band corresponding to actin was excised and digested with trypsin. The peptides were analyzed by nanospray mass spectrometry. The intensities of a pair of light and heavy versions of a single actin peptide are shown.

A B C