Supporting Information. for. Development and Application of a Quantitative Multiplexed Small GTPase Activity. Assay Using Targeted Proteomics

Transcription

1 Supporting Information for Development and Application of a Quantitative Multiplexed Small GTPase Activity Assay Using Targeted Proteomics Cheng-Cheng Zhang, 1 Ru Li, 1,2 Honghui Jiang, 1,2 Shujun Lin 1, Jason C. Rogalski 3, Kate Liu 1 1, 2, 4*, Juergen Kast 1. The Biomedical Research Centre, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada 2. Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1, Canada 3. The Centre for High-throughput Biology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada 4. The Centre for Blood Research, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada * Telephone: 1-(604) Fax: 1-(604) juergen@brc.ubc.ca. 1

2 Supplementary Table 1. Different small GTPase isoforms can be precipitated by effector binding domains in human platelets. Platelet lysate (500μg) was treated with GTPγs to activate small GTPases, which were then precipitated by an effector binding domain bound to glutathione beads. The bound small GTPases were then eluted and run on a 12% SDS-PAGE, where the kda region was excised and in-gel digested. Digested samples were analyzed by FT-ICR mass spectrometer. Raw data is accessible at gpmdb.thegpm.org with GPMDB model numbers. Effector binding Domain GPMDB model number Small GTPase Rap1B GST-RalGDS-RBD GPM Rap1A Rap2B Cdc42 GST-PAK1-PBD GPM Rac2 Rac1 GST-Rhotekin-RBD GPM RhoA RhoC GST-Raf1-RBD GPM GPM NRas/HRas KRas 2

3 Small GTPase NRas HRas KRas RhoA RhoB RhoC Rap1A Rap1B Rac1 Rac2 Supplementary Table 2. Candidate proteotypic peptides for small GTPase isoforms. These peptides passed the following filters: unique to small GTPase isoform, peptide length of 8-25 amino acids, maximum one chemically-induced modification for cysteine and/or methionine, tryptic peptide with maximum one miss cleavage, and minimum one observation in the GPMDB. * represents the peptides that were synthesized and further tested. # denotes number of GPMDB observations as of May 31, Cys, cysteine; Met, methionine; ESS, empirical suitability score from peptide atlas. Candidate proteotypic peptides Length Modifi-cation Miss cleavage GPMDB observations z=1 z=2 z=3 SFADINLYR* 9 No TGEGFLCVFAINNSK* 15 Cys No SFEDIHQYR* 9 No SYGIPYIETSAK* 12 No SFEDIHHYR* 9 No DSEDVPMVLVGNK* 13 Met No VKDSEDVPMVLVGNK* 15 Met Yes TRQGVDDAFYTLVR 14 Yes DQFPEVYVPTVFENYVADIEVDGK* 24 No IGAFGYMECSAK* 12 Cys, Met No MKQEPVKPEEGR 12 Met Yes DGVREVFEMATR 12 Met Yes IQAYDYLECSAK* 12 Cys No HFCPNVPIILVANK* 14 Cys No LVVVGDGACGK 11 Cys No HFCPNVPIILVANKK 15 Cys Yes KLVVVGDGACGK 12 Cys Yes DQFPEVYVPTVFENYIADIEVDGK* 24 No ISAFGYLECSAK* 12 Cys No EGVREVFEMATR 12 Met Yes DTEDVPMILVGNK* 13 Met No VKDTEDVPMILVGNK* 15 Met Yes DTDDVPMILVGNK* 13 Met No VKDTDDVPMILVGNK* 15 Met Yes HHCPNTPIILVGTK* 14 Cys No LTPITYPQGLAMAK* 14 Met No KLTPITYPQGLAMAK* 15 Met Yes EIGAVKYLECSALTQR 16 Cys Yes LAPITYPQGLALAK* 14 No HHCPSTPIILVGTK* 14 Cys No AVLCPQPTR 9 Cys No ESS 3

4 Rap2B Cdc42 KLAPITYPQGLALAK 15 Yes EIDSVKYLECSALTQR 16 Cys Yes ASVDELFAEIVR* 12 No SALTVQFVTGSFIEK* 15 No ALAEEWSCPFMETSAK 16 Cys, Met No VDLEGEREVSYGEGK 15 Yes NKASVDELFAEIVR 14 Yes VPMILVGNKVDLEGER 16 Met Yes TPFLLVGTQIDLR* 13 No WVPEITHHCPK* 11 Cys No TCLLISYTTNK* 11 Cys No DDPSTIEK 8 No

5 Supplementary Table 3. Technical replicates of activity levels of small GTPases in platelets treated with GTPγs. Platelet lysates were treated with GTPγs for 15 min, and then lysed, and the multiplexed active small GTPase pull down assay was applied. All ten measurements for the quantifiable proteotypic peptides had a CV < 20%, and nine of them had a CV < 15%. SD, standard deviation; CV, coefficient of variation, C*, cysteine carbamidomethylation (n = 5). Small Proteotypic peptide Light/Heavy peak area ratio GTPase sequence Mean SD CV% NRas SFADINLYR HRas SYGIPYIETSAK KRas VKDSEDVPMVLVGNK RhoA IGAFGYMEC*SAK RhoB IQAYDYLEC*SAK RhoC ISAFGYLEC*SAK Rap1A VKDTEDVPMILVGNK Rap1B VKDTDDVPMILVGNK Rap2B ASVDELFAEIVR Rac1 LTPITYPQGLAMAK Rac2 LAPITYPQGLALAK Cdc42 TC*LLISYTTNK

6 Supplementary Table 4. Technical replicates of activity levels of small GTPases in platelets treated with thrombin. Platelets were stimulated with 0.2 U ml -1 thrombin for 1 min, and then lysed, and the multiplexed active small GTPase pull down assay was applied. Activation levels of platelets are reported relative to the unstimulated platelets (average of three measurements) which was normalized to 100%. All quantitative measurements for the thrombin-treated samples had a CV < 20%, and 90% (9/10) had a CV < 15%. SD, standard deviation; CV, coefficient of variation (n = 3). Small GTPase Control (%) Thrombin (%) SD CV% NRas KRas RhoA RhoC Rap1A Rap1B Rap2B Rac Rac Cdc

7 Supplementary Figure 1. Validation of activity of effector binding domains using Western blot. (a) Active Rap1 pull down by GST-RalGDS-RBD, (b) active Ras pull down by GST-Raf1-RBD, (c) active Cdc42 pull down by GST-PAK1-PBD, and (d) active Rho pull down by GST-Rhotekin-RBD. GTPγs and GDP were used as positive and negative controls, respectively. For sample loading, 25 μl platelet lysate was used for western blot control, 250 μl platelet lysate was used for pull down experiment, 25 out of 500 μl of the sample flow through was used for western blot. Protein level was detected using an Odyssey infrared imaging system. FT, flow through. 7

8 Supplementary Figure 2. Sequence alignment for various small GTPases reveals high similarity. Sequence alignment was generated using the Clustal Omega. Candidate proteotypic peptides using relaxed selection criteria are highlighted. 8

9 Supplementary Figure 3. Top transition selection and peptide optimization using triple quadrupole mass spectrometry. NRas proteotypic peptide SFADINLYR with heavy labeled arginine ( 13 C 6 ; 15 N 4 ) was used as an example. (a) Top three transitions were selected based on intensity; (b) Optimal FV for precursor ion m / z was achieved at 200 V (FV range = 60 to 280 V with 20 V increment tested); (c) Initial CE optimization for transition m / z CE = 15 V (CE range = 5 to 35 V with 5 V increment tested); (d) Optimal CE was achieved at 13 V for the same transition (CE range = 11 to 19 V with 1 V increment tested). FV, fragmentor voltage; CE, collision energy. 9

10 Supplementary Figure 4. Validation of MRM assay using Western blot. Active Rap1B pull down experiments were performed in platelets stimulated with positive control (GTPγs), 1 U ml -1 thrombin, 0.19 mg ml -1 collagen or 20 µm LPA for 10 min, and analyzed by (a) MRM assay and (b) Western blot. Three transitions, i.e. y4, y5 and y8, of VKDTDDVPMILVGNK for Rap1B were used for quantification. The coefficient of variation for the three transitions at each condition was less than 5%. 10

11 Supplementary Figure 5. Time-resolved activation profiles of (a) Rap1A and (b) Rap1B (proteotypic peptides with methionine oxidation) in platelets stimulated with different agonists. Platelets were stimulated with 0.2 U ml -1 thrombin (Thr), 100 µm ADP, or 20 µm LPA at pre-defined time points, the platelets were then lysed, and the multiplexed active small GTPase pull down assay was applied. Activation levels of platelets are reported relative to the positive control (treatment of platelets with GTPγs) which was normalized to 100% (n=3). 11