Supporting Information

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1 Supporting Information Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres to Achieve Automated Proteome Quantification Huiming Yuan #, Shen Zhang #, Baofeng Zhao, Yejing Weng, Xudong Zhu, Senwu Li, Lihua Zhang*,Yukui Zhang CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian , China; University of Chinese Academy of Sciences, Beijing , China. # These authors contributed equally to this work * To whom correspondence should be addressed. Prof. Lihua Zhang: phone, ; fax, ; , lihuazhang@dicp.ac.cn. Table of Contents Materials and Reagents...S-3 Protein extraction...s-3 Preparation of GO@PEI@polymer particles...s-4 Characterization of GO@PEI@polymer particles...s-4 Trypsin immobilization...s-4 Evaluation of the peptide carryover on the IMER...S-5 Trypsin digestion and 18 O labeling...s-5 Life time of the IMER......S-6 MALDI-TOF MS detection... S-6 Data analysis...s-6 References...S-7 Table S-1...S-8 Table S-2...S-8 S-1

2 Table S-3...S-9 Figure S-1... S-10 Figure S-2...S-11 Figure S-3...S-11 Figure S-4...S-13 Figure S-5...S-14 Figure S-6...S-15 Figure S-7...S-16 Figure S-8...S-17 S-2

3 Materials and Reagents Bovine serum albumin (BSA, bovine serum), Trypsin (bovine pancreas, TPCK treated), formic acid (FA), α-cyano-4-hydroxycinnamic acid (CHCA), polyethylenimine (average Mw=800, PEI), and protease inhibitor cocktail were ordered from Sigma Aldrich (St. Louis, MO, USA). Dithiothreitol (DTT) and iodoacetamide (IAA) were obtained from Acros (Morris Plains, NJ, USA). Acetonitrile (ACN, HPLC grade) was purchased from Merck (Darmstadt, Germany). Water was purified by a Milli-Q system (Millipore, Milford, MA, USA). 18 O-Water (97%) was obtained from the Shanghai Research Institute of Chemical Industry (Shanghai, China). All other chemicals and solvents were of analytical grade. A precise syringe pump (Baoding Longer Pump Company, Baoding, China) was used to pump the protein solution through the IMERs. A BCA protein assay kit was obtained from the Beyotime Institute of Biotechnology (Tianjin, China). Acrylic polymer particles with amine groups (10 μm, 1,000 Å) were obtained from Suzhou Nanomicro Technology Inc. (Suzhou, China). Fused-silica capillaries (250 µm i.d. 375 µm o.d.) were obtained from Sino Sumtech (Handan, China). Protein extraction E. coli cells were cultured in LB medium at 37 C for 24 h. The cells were harvested and then washed with ice-cold PBS 3 times, finally suspended in an extraction buffer composed of 8 M urea and 1% (v/v) protease inhibitor cocktail. The suspension was ultrasonicated for 100 s at 100 W and centrifuged at 20,000 g for 30 min. Then, the supernatant was collected as the soluble fraction of E. coli cell lysate. Hepatocarcinoma ascites syngeneic cells with high (Hca-F) and low (Hca-P) lymph node metastasis rates were donated by Professor Shujuan Shao (Dalian Medical University). The grown cells were suspended in an extraction buffer composed of 8 M urea and 1% (v/v) protease inhibitor cocktail, followed by ultrasonication for 100 s at 100 W and centrifugation at 25,000 g for 40 min. The supernatant was collected as the soluble fraction proteins. The supernatants from E.coil, Hca-F, and Hca-P cells were precipitated by the addition S-3

4 of ice-cold acetone. After centrifugation, the pellets were lyophilized by a Speed Vac Concentrator (Thermo Fisher Scientific, CA) and re-dissolved in 50 mm NH 4 HCO 3 (ph 7.8). For 18 O labeling, the NH 4 HCO 3 buffer was made with 18 O-water instead of 16 O-water. The protein concentration was determined by a BCA assay. Preparation of GO@PEI@polymer particles Acrylic polymer particles with amino groups (150 mg, 10 μ, 1,000 Å) were suspended in phosphate buffer (PB, ph 8.0) containing 5% glutaraldehyde (v/v) and vortexed for 3 h at room temperature. After centrifugation, the modified particles were obtained, and the unreacted glutaraldhyde was removed by flushing the particles with ethyl alcohol and water. Then, 5 mg/ml PEI was added to the suspension of the modified particles and reacted for 3 h at room temperature. After centrifugation, PEI-modified polymer particles were obtained, and the unreacted PEI was removed by washing the PEI-modified particles with ethyl alcohol and water. Finally, approximately 75 mg of PEI-modified particles was mixed with 1.5 ml (1 mg/ml) of GO suspension and vortexed for 6 h at room temperature. After centrifugation, the obtained GO@PEI@polymer particles were washed with water, ethyl alcohol, and phosphate buffer. Characterization of GO@PEI@polymer particles Dynamic light scattering (DLS) and zeta potential measurements were performed on a Malvern Nano Z Zetasizer (Worcestershire, UK). The particles were dispersed in water during the measurements. The chemical composition was performed on an X-ray photoelectron spectrometer (XPS, Thermo ESCALAB 250 Xi) with Al Kα radiation as the X-ray source (Thermo, Waltham, MA, USA). Transmission electron microscopy (TEM) analysis was performed on a JEM-2000EX instrument (120 kv, Tokyo, Japan). Trypsin immobilization The GO@PEI@polymer particles were packed into a 250 μm capillary tube; then, a 2 mg/ml trypsin solution containing 50 mm benzamidine was pumped into the capillary tube S-4

5 for 24 h at 4 C. The obtained IMER was flushed by 50 mm ammonium bicarbonate (ph 8.0) to remove the residual trypsin; the unreacted residual trypsin was collected and analyzed by a BCA assay. The IMER was stored at 4 C for further use. For comparison, amino-functionalized polymer microparticles and PEI-modified polymer microparticles were packed into capillary tubes with the same dimensions. The supports were activated by flushing a solution of 5% (v/v) glutaraldehyde for 6 h at room temperature, and trypsin was covalently bonded by continuously pumping 3 mg/ml trypsin dissolved in 100 mm phosphate buffer (ph 8.0) containing 50 mm benzamidine and 5 mg/ml sodium cyanoborohydride for 24 h at 4 C. After being purged with 1 M Tris-HCl (ph 8.0) and 20% acetonitrile (ACN) for 4 h, the IMERs were filled with 0.02% (w/v) NaN3 solution and stored at 4 C before use. Evaluation of the peptide carryover on the IMER The adsorption of peptides on the IMER based on GO@PEI@polymer particles was evaluated by the following steps. The IMER was flushed in sequence with light and heavy formaldehyde-labeled peptides from E. coli (0.1 mg/ml) at a flow rate of 1 μl/min for 10 min. The light fraction was discarded, whereas the heavy fraction was collected and subjected to nano-lc-ms/ms analysis. Trypsin digestion and 18 O labeling For traditional in-solution protocols, BSA and E. coli extracts were digested by trypsin with a substrate-to-enzyme ratio of 25:1(m/m), which was followed by incubation at 37 C for 12 h. After being desalted, the tryptic digests from BSA and E. coli extracts were lyophilized by a Speed Vac Concentrator and re-suspended in trypsin (25:1, m/m) prepared with 100 mm ammonium acetate buffer (dissolved in 18 O-water, ph 6.0). After incubation for 24 h at 37 C, the protein solutions were boiled for 10 min at 100 C to deactivate residual trypsin [1]. For the IMER, BSA and E. coli extracts dissolved in NH 4 HCO 3 buffer (prepared with 18 O-water) were pumped into the IMERs at a flow rate of 1 μl/min at 37 C, and the digests were collected for further MS analysis. Furthermore, in our experiments, we added 16 O-water to redissolve 18 O labeled peptides obtained by the IMER, and kept them at 4 C for a week. The peptides were analyzed by MALDI-TOF MS. S-5

6 Lifetime of the IMER The lifetime of the IMER was evaluated by digestion and 18 O labeling of 0.1 mg/ml BSA at the flow rate of 1 μl/min in consecutive 10 days (100 runs). The collected digests were analyzed by MALDI-TOF MS. MALDI-TOF MS detection MALDI-TOF MS experiments were performed on a Bruker ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) using a Smartbeam laser as an ionization source. With a polished target, 7 mg/ml CHCA dissolved in a 2:1 mixture of ACN/H 2 O containing 0.1% (v/v) TFA was used as the matrix. MALDI-TOF MS experiments were performed with constant laser intensity in the positive ion mode. All mass spectra were obtained with an accumulation of 800 laser shots in the reflector mode with an accelerating voltage of kv. Data analysis For MALDI-TOF MS, several peptides within a broad range of m/z were chosen to calculate the 18 O labeling efficiency from the MALDI data in BSA digests, and the efficiency of incorporation of one or two 18 O atoms was manually calculated according to the following equations [2]: (1) The theoretical intensities of Mi were calculated from its sequence according to the MS-isotope value obtained from the ProteinProspector website ( (2) For LC-MS analysis, the acquired raw files were analyzed with MaxQuant (version S-6

7 ). Cysteine carbamidomethylation was searched as a fixed modification, whereas N-terminal acetylation and methionine oxidation were searched as variable modifications. Enzyme specificity was set as trypsin. Two missed cleavages were allowed, and at least six amino acids were required per identified peptide. For 18 O based quantitative analysis, 18 O was set as the heavy label. References (1) Yang, S. J.; Nie, A. Y.; Zhang, L.; Yan, G. Q.; Yao, J.; Xie, L. Q.; Lu, H. J.; Yang, P. Y. Journal of Proteomics, 2012, 75, (2) López-Ferrer, D.; Ramos-Fernandez, A.; Martinez-Bartolome, S.; Garcia-Ruiz, P.; Vázquez, J. Proteomics, 2006, 6, 4-6. S-7

8 Table S-1. Zeta potentials of GO, polymer particles, particles and particles determined by dynamic light scattering. Zeta potential GO Polymer particle particle ymer particle (mv) / Average Table S-2. Elemental analysis of polymer particles and PEI@polymer particles by X-ray photoelectron spectroscopy (XPS). Atomic PEI@polymer Polymer particle % particle C1s N1s O1s S-8

9 Table S-3. Comparison of the labeling efficiencies obtained by the IMER and a traditional off-line protocol. In the experiments, the 8 most intense peaks from the MALDI-TOF MS spectra were selected. Sequence m/z Labeling efficiency (%) IMER (2.5 min) Labeling efficiency (%) off-line protocol (38 h) K.YLYEIAR.R K.CCTESLVNR R R.FKDLGEEH FK.G K.LGEYGFQN ALIVR.Y K.LKPDPNTL CDEFK.A R.KVPQVSTPT LVEVSR.S K.DAFLGSFLY EYSR.R R.RPCFSALTP DETYVPK.A Average labeling efficiency (%) S-9

10 Figure S-1. TEM morphologies of a) polymer microparticle b) GO, c) hybrid particles ( 4,000), and d) GO@PEI@polymer particles ( 15,000). S-10

11 Figure S-2. MALDI-TOF MS spectra of BSA digests by the IMER in H 2 18 O (a) and H 2 16 O (b). S-11

12 (1) (2) (3) (4) (1) (2) (3) (4) Figure S-3. Comparison of peptide labeling efficiencies obtained by the IMER based on particles (2), particles (3), and polymer particles (4). In the experiments, the same protein digestion was also performed in H 16 2 O (1).The sequences of selected peptides were as follows: a) K.YLYEIAR.R; b) K.VLTSSARQR.L; c) K.LGEYGFQNALIVR.Y; d) R.KVPQVSTPTLVEVSR.S; e) K.DAFLGSFLYEYSR.R; and f) R.RPCFSALTPDETYVPK.A. S-12

13 Figure S4. Evaluation of protein digestion by upimer in proteome quantification. a). The log2 (H/L) ratio distribution of peptides from E. coli samples with and without missed cleavage sites. b). The observed log2 (H/L) ratios of the quantified proteins in triplicate runs. S-13

14 Figure S-5. Carryover of peptides on the IMER. Three representative peptides are listed, and the sequences of these peptides were as follows: a) GIISVEQEIK; b) AIEEANADIEVK; and c) ISGSVTVGETPVIR. S-14

15 Figure S-6. Comparison of the labeling efficiencies for 18 O-peptides produced by the IMER in 18 O-water (above) and re-dissolved in 16 O-water for 7 days (below). The sequences of the selected peptides were as follows: a) K.VLTSSARQR.L; b) K.LGEYGFQNALIVR.Y; c) R.KVPQVSTPTLVEVSR.S; d) K.DAFLGSFLYEYSR.R; e) K.YNGVFQECCQAEDK.G; and f) R.RPCFSALTPDETYVPK.A. S-15

16 Figure S-7. Evaluation of durability of the IMER. a) The sequence coverage of BSA achieved by upimer in consecutive 100 runs (10 days); b) The 18 O labeling efficiency achieved by upimer in consecutive 100 runs (10 days) S-16

17 Figure S-8. Differential proteomics analysis of Hca-F and Hca-P. a) Volcano plot of the gobal quantification of proteins in Hca-F/P cell line; b) Gene ontology analysis of quantified proteins. S-17