SH2 superbinder modified monolithic capillary column for the sensitive analysis of protein tyrosine phosphorylation Yating Yao 1,2,4, Yangyang Bian 1,3,4, Mingming Dong 1,5,*, Yan Wang 1,2, Jiawen Lv 1,2, Lianfang Chen 1,2, Hongwei Wang 1,2, Jiawei Mao 1,2, Jing Dong 1, Mingliang Ye 1,* 1 CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China; 2 University of Chinese Academy of Sciences, Beijing 100049, China; 3 Medical Research Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, Henan 450052, China; 5 Dalian Ocean University, Dalian 116023, China; 4 These authors contributed equally to the work. * To whom correspondence should be addressed: *Phone: +86-411-84379610. Fax: +86-411-84379620. E-mail: mingliang@dicp.ac.cn. (M.L. Ye) *Phone: +86-411-84379620. Fax: +86-411-84379620. E-mail: dongmm@dicp.ac.cn. (M.M. Dong) Supporting Information Figure S1. Scheme of immobilizing SH2 superbinder on the poly (glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column. Figure S2. Motif enrichment analysis for the ptyr sites identified from the Hela samples with different amounts of starting materials. Figure S3. Venn diagrams illustrating the unique ptyr sites found in SH2 microreactor method and SH2 sepharose method. Figure S4. Protein abundance distribution of the identified tyrosine phosphorylated proteins in SH2 microreactor enrichment method and SH2 sepharose beads enrichment method with 100 µg starting materials. Figure S5. Overlap of ptyr site identifications obtained from four SH2 microreactors in parallel. Table S1. Summary of the identifications obtained by the SH2 microreactor enrichment method and SH2 sepharose beads enrichment method. S-1
Figure S1. Scheme of immobilizing SH2 superbinder on the poly (glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column. S-2
Figure S2. Motif enrichment analysis for the ptyr sites identified from the Hela samples with different amounts of starting materials (1000 µg, a and e, 500 µg, b and f, 200 µg, c and g, 100 µg, d and h) using SH2 microreactor (a-d) and SH2 sepharose beads (e-h). Two replicate LC MS/MS runs were performed, and the results were combined. S-3
Figure S3. Venn diagrams illustrating the unique ptyr sites found in SH2 microreactor method and SH2 sepharose method with (a) 100 µg and (b) 1000 µg starting materials. Two replicate LC MS/MS runs were performed, and the results were combined. All sites have a Localization Probability 0.75 and Score Difference 5. S-4
Figure S4. Protein abundance distribution of the identified tyrosine phosphorylated proteins in SH2 microreactor enrichment method and SH2 sepharose beads enrichment method with 100 µg starting materials. S-5
Figure S5. Overlap of ptyr site identifications obtained from four SH2 microreactors in parallel. S-6
Table S1. Summary of the identifications obtained by the SH2 microreactor enrichment method and SH2 sepharose beads enrichment method. Different amounts of starting materials were respectively enriched by the indicated two methods. Two replicate LC MS/MS runs were performed, and the results were combined. All sites have a Localization Probability 0.75 and Score Difference 5. S-7
Expression, Purification, and Immobilization of the SH2 Superbinder SH2 superbinder was expressed as hexahistidine-tagged proteins in E. coli BL21 (DE3) as previous reported(1), and purified on Ni-NTA beads according to the manufacturer s instructions. For the experiments where SH2 sepharose beads were used, the superbinder was immobilized on CNBr-activated Sepharose 4B (GE Healthcare) following the manufacturer s instructions, and the final concentration for the immobilized SH2 superbinder was 1 mg of protein per ml of medium. Shc1 immunoprecipitation and on-beads digestion The Shc1 complex preparation was according to Zheng s protocols with some modifications(2). Briefly, HeLa cell lines expressing Shc1 were grown in DMEM plus 10% FBS. Before harvesting, the cells were treated with 2 min EGF stimulation, and then immediately washed with ice-cold PBS three times to quench cell signaling. Subsequently, the cells were lysed in NP-40 lysis buffer containing 50 mm HEPES, 150 mm NaCl, 2.5 mm MgCl 2, 1 mm DTT, 1 mm NaF, 1 mm Na 3 VO 4, 1 mm β-glycerolphosphate, 10 mm Na 4 P 2 O 7, 10% glycerol (v/v) and 2% cocktail (v/v) at ph 8.0. The cell lysates were centrifuged at 20,800 g for 20 minutes and the supernatants were collected. Shc1 complex was co-immunoprecipitated by incubating the lysates with anti-flag M2 antibody-conjugated sepharose at 4 o C overnight. The sepharose were washed three times with NP-40 lysis buffer and two times with 20 mm ammonium bicarbonate. Then the beads were resuspended in 20 µl ammonium bicarbonate with the addition of trypsin (enzyme :protein ~ 1:50). The digestion was performed at 37 o C overnight and stopped by adding 3% FA to the reaction. Finally, the supernatants were collected and dried for further analysis. S-8
References 1. Bian, Y.; Li, L.; Dong, M.; Liu, X.; Kaneko, T.; Cheng, K.; Liu, H.; Voss, C.; Cao, X.; Wang, Y.; Litchfield, D.; Ye, M.; Li, S. S.; Zou, H., Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder. Nat Chem Biol 2016, 12, (11), 959-966. 2. Zheng, Y.; Zhang, C.; Croucher, D. R.; Soliman, M. A.; St-Denis, N.; Pasculescu, A.; Taylor, L.; Tate, S. A.; Hardy, W. R.; Colwill, K.; Dai, A. Y.; Bagshaw, R.; Dennis, J. W.; Gingras, A. C.; Daly, R. J.; Pawson, T., Temporal regulation of EGF signalling networks by the scaffold protein Shc1. Nature 2013, 499, (7457), 166-71. S-9