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 day3 H H L L day2 L LH day1 day0 full MS scan (negative mode) full MS scan (positive mode) fragmentation of m/z 867.57 (positive mode) RPHFPQFpSYSASGTA 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. 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. L/H=1/2L/H=1/2 L/H=1/1L/H=1/1 L/H=2/1L/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