Abstract

1
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
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 13Carginine 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.
A
B
B
A
C
L/H1/2
L/H1/1
L/H2/1
C
D
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.
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
11, panel A 21, panel B or 12, 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.
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.
Method
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.
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.
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.
Results

• 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.

• 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.

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 13Carginine (day 1-day3).