An evolutionary view on thylakoid protein phosphorylation uncovers ...
Abstract An important goal of the AfCS Protein Chemistry Laboratory is the analysis of...
1
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 13 C(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 [ 13 C]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 13 C-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 [ 13 C]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 Peptides Shu 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 +TO F M S :1.817 to 7.818 m in from hs040414_S ILA C _P rotein_467_D ay3_3A .w iff a=3.58510353443061910e-004, t0=3.20920165373026980e+001 M a x. 567 .3 cou nts. 755.0 756.0 75 7.0 758.0 759.0 7 60.0 76 1.0 762 .0 76 3.0 7 64 .0 76 5.0 m /z,am u 0 5 10 15 20 25 30 35 40 42 760.6319 761.1297 761.6246 762.1236 757.5946 763.5809 755.5774 764.5798 758.5864 756.5751 759.5683 760.1281 763.0774 756.0610 day3 H +TO F M S : 1.334 to 7 .81 8 m in fro m hs040414_S ILA C _P rotein_467_D ay2_2A .w iff a=3.58510353443061910e-004,t0=3.20920165373026980e+001 M ax.951.6 counts. 75 5.0 75 6.0 75 7.0 758.0 759.0 760 .0 7 61.0 762.0 763.0 7 64.0 76 5.0 m /z, am u 0.0 2.0 4.0 6.0 8.0 1 0.0 1 2.0 1 4.0 1 6.0 1 8.0 2 0.0 2 2.0 2 4.0 2 5.2 760.6201 761.1215 761.6160 757.6033 758.1087 764.0547 758.5869 755.5809 762.0987 765.0367 763.5799 756.5613 759.5730 760.0842 763.0434 759.0714 H L L day2 +TO F M S :0.817 to 5.018 m in from S am ple 2 (P o sitive M S ) ofhs040414_S ILA C _ P ro tein _467_ D ay1... a=3.58510353443061910e-004,t0=3.20920165373026980e+001 M ax.514.6 counts. 755.0 7 56 .0 757.0 7 58 .0 759.0 760.0 761.0 7 62.0 76 3.0 76 4.0 76 5.0 m /z,am u 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 760.6151 757.6045 761.1203 758.1092 761.6098 758.6022 755.5810 764.5780 763.5638 759.0533 762.0913 764.0617 756.5557 759.5726 755.0444 +TO F M S : 0.7 67 to 4 .30 1 m in from hs040414_S ILA C _P rotein_467_D ay0_0A .w iff a=3.58510353443061910e-004,t0=3.20920165373026980e+001 M ax.270.1 counts. 7 55.0 75 6.0 757.0 75 8.0 7 59.0 76 0.0 761.0 76 2.0 763.0 76 4.0 765.0 m /z, am u 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 0.0 1 1.0 1 2.0 1 3.0 1 4.0 1 5.0 1 6.0 1 7.0 1 8.0 757.6151 758.1204 758.6155 755.5783 760.5935 761.5872 759.5942 764.5780 756.5713 762.5709 759.1287 763.5749 755.0874 764.1196 756.0558 L L H day1 day0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m /z,am u 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 635.1837 643.1786 681.2843 8 28 .3 78 6 899.7992 864.7202 8 18 .8 88 5 964.3430 761.2618 890.5584 601.1922 819.5530 1228.8401 973.3439 588.1837 1271.3968 486.1625 895.8882 1058.8050 664.2337 329.1596 951.2689 1176.3417 1344.3488 607.2034 400 500 600 700 800 900 1000 1100 1200 1300 m /z,am u 0 100 200 300 400 500 600 700 800 900 1000 1100 637.4326 630.5546 415.8580 601.7700 645.4383 645.9389 416.3605 821.2058 590.4307 545.7099 517.4561 830.7107 376.2806 707.4813 867.5723 426.8530 1073.7392 615.4291 896.6338 1273.9071 1231.3157 455.8422 804.5474 100 200 300 400 500 600 700 800 900 1000 1100 1200 m /z,am u 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 254.2307 538.4286 818.0975 510.4263 373.3084 809.0869 763.5776 391.3191 606.4451 490.3730 195.1676 173.1449 230.1769 867.0894 773.5589 694.5090 388.2881 1097.7482 520.3973 246.1771 493.3773 237.2022 997.7566 726.4972 849.0863 147.1672 full MS scan (negative mode) full MS scan (positive mode) fragmentation of m/z 867.57 (positive mode) RPHFPQFpS YSASGTA 700 750 800 850 900 950 1000 1050 1100 1150 1200 m /z,am u 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 89 865.0543 905.2512 731.9238 785.1818 985.9125 1098.2667 1178.3834 710.3400 876.2714 867.4667 893.3375 1046.6750 Precursor scan (negative mode) phosphopeptide at m/z=865.72 m/z 864.72 (M) -2 phosphopeptide at m/z=865.05 (M) -2 phosphopeptide at at 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. -T O F M S :0.067 to 1.034 m in fro m S am p le 3 (N e g a tive M S ) ofhs040519_P roteinLab_497_A kt1_C L... a=3.58623901186383460e-004,t0=6.39034864398807510e+001 M ax.252.7 counts. 86 1 .0 8 6 2.0 863.0 86 4 .0 8 65 .0 8 6 6.0 867.0 86 8 .0 8 69 .0 8 7 0.0 871.0 87 2 .0 8 73 .0 8 7 4.0 875.0 m /z,am u 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 8 6 9 .8 41 9 864.8276 865.3320 870.3496 8 6 5.8 3 3 0 8 7 0 .8 4 59 86 6 .3 3 6 6 869.3517 860.4393 871.3758 863.3722 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. +TO F M S :7.018 to 10.886 m in from hs040423_SILAC_Protein_482_2D_L-H_1-2.wiff a=3.58510353443061910e-004,t0=3.20920165373026980e+001 M ax.805.4 counts. 74 8.0 749.0 750.0 751.0 75 2.0 75 3.0 754 .0 755 .0 756 .0 75 7.0 75 8.0 m /z,am u 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 753.5649 754.0681 750.5583 +TO F M S :19.288 to 25.339 m in from hs040423_SILAC_Protein_482_2E_L-H_2-1.wiff a=3.58510353443061910e-004,t0=3.20920165373026980e+001 M ax.288.0 count 74 8.0 74 9.0 75 0.0 75 1.0 752.0 753.0 754 .0 75 5.0 75 6.0 75 7.0 7 58.0 m /z,am u 0 20 40 60 80 100 120 140 160 180 200 211 750.5759 751.0782 753.5872 751.5788 754.0873 754.5905 752.0809 L/H=1/2 +T O F M S :3.40 1 to 8 .9 52 m in from hs040423_SILAC_Protein_482_2C_L-H_1-1.wiff a=3.58510353443061910e-004, t0=3.20920165373026980e+001 M ax.1615.5 counts. 748.0 7 49.0 75 0.0 751.0 752.0 7 53.0 75 4.0 755.0 756.0 75 7.0 75 8.0 m /z,am u 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 99 750.5531 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
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Transcript of Abstract An important goal of the AfCS Protein Chemistry Laboratory is the analysis of...
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
+TOF MS: 1.334 to 7.818 min from hs040414_SILAC_Protein_467_Day2_2A.wiffa=3.58510353443061910e-004, t0=3.20920165373026980e+001
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
8
10
12
14
16
18
20
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
+TOF MS: 0.767 to 4.301 min from hs040414_SILAC_Protein_467_Day0_0A.wiffa=3.58510353443061910e-004, t0=3.20920165373026980e+001
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
150
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
600
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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20
25
30
35
40
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
20
30
40
50
60
70
80
90
100
110
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
15
20
25
30
35
40
45
50
55
60
65
70
75
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
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