Supporting information

Transcription

1 Supporting information Simple and Integrated Spintip-based Technology Applied for Deep Proteome Profiling Wendong Chen,, Shuai Wang, Subash Adhikari, Zuhui Deng, Lingjue Wang, Lan Chen, Mi Ke, Pengyuan Yang, and Ruijun Tian *,, Department of Chemistry and Shenzhen Key Laboratory of Cell Microenvironment, South University of Science and Technology of China, Shenzhen , China Department of Chemistry, Fudan University, Shanghai , China ENT Institute of Shenzhen University, Shenzhen Longgang ENT Hospital, Shenzhen , China * tianrj@sustc.edu.cn. Phone: Page S-1

2 Contents 1. Experimental methods. 2. Figure S-1. Testing the binding capacity of SCX beads in the SISPROT by SDS-PAGE analysis (Coomassie brilliant blue staining) of the BSA flow through (FT). 3. Figure S-2. Common and unique proteins identified with a minimum of two peptides per protein from 20,000 HEK 293T cells in triplicate analysis. 4. Figure S-3. Label-free quantification (LFQ) analysis of 100,000 SHED cells as processed by the SISPROT. 5. Table S-1. Cells counted by a hemocytometer. 6. Table S-2. Peptides and proteins identified from 20,000 HEK 293T cells in triplicate analysis. 7. Table S-3. Label-free quantification analysis of 20,000 HEK 293T cells by MaxQuant (Excel file). 8. Table S-4. Peptides and proteins identified from 100,000 SHED cells in triplicate analysis. 9. Table S-5. The identified proteins and the distribution of SHED protein abundance as annotated by copy number per cell (Excel file). 10. Table S-6. GO cellular component of each protein for SHED (Excel file). 11. Table S-7. GO cellular component of each membrane protein for SHED (Excel file). 12. Table S-8. Label-free quantification analysis of 100,000 SHED cells by MaxQuant (Excel file). Page S-2

3 Experimental methods Protocol of the SISPROT 1. Materials Solutions and Reagents Milli-Q water (H 2 O) Methanol (chromatographic grade) PCB buffer: 10 mm potassium citrate in H 2 O, ph 3 TFA solution: 2% (v/v) trifluoroacetic acid in H 2 O Wash buffer: 20% (v/v) acetonitrile, 8 mm potassium citrate in H 2 O, ph 3 Reduction buffer: 10 mm TCEP, 9 mm potassium citrate in H 2 O, ph 3, prepare on the day of experiment Digestion buffer: 2 µg/µl trypsin (V5111, Promega) in 10 mm iodoacetamide, 100 mm Tris-HCl, ph 8, prepare during the reduction step and keep on ice in darkness Transfer buffer: 200 mm ammonium formate in H 2 O, ph 10 Desalting buffer: 5 mm ammonium formate in H 2 O, ph 10 Elution buffer: 80% (v/v) acetonitrile, 5 mm ammonium formate in H 2 O, ph 10 Fractionation buffer: 3%, 6%, 9%, 15%, and 80% (v/v) acetonitrile, 5 mm ammonium formate in H 2 O, ph 10 Resuspension solution: 0.1% (v/v) formic acid in H 2 O Equipment Bench-top centrifuge (e.g. Centrifuge 5424, Eppendorf) Plastic syringe, 20 ml with a custom adapter to fit into 200 µl pipette tip Vacuum concentrator (e.g. CentriVap Concentrator, LABCONCO) SISPROT tip: Firstly, pack one to five plugs of Empore C 18 (3M) membrane into a 200 µl pipette tip. Then, prepare strong cation exchange (SCX) slurry by suspend 10 mg of 20 µm POROS SCX beads (Applied Biosystems, USA) in 1 ml water and introduce the SCX beads into the pipette tip by centrifuging at the speed of 2000 g. Page S-3

4 Typically, one plug of C18 membrane is used for 5 µg of digested peptides and mg SCX beads is used for 2-20 µg proteins (the binding capacity of SCX beads is ~30 µg proteins/mg). 2. Procedure: (1) Wash the SISPROT tip with 40 µl of methanol. Centrifuge at 1500 g for 0.5 min or until all liquid is passed through. (2) Equilibrate the SISPROT tip with 40 µl of PCB buffer. Centrifuge at 1500 g for 1 min or until all liquid is passed through. (3) Acidify the sample with TFA solution to ph 2-3. Load the sample onto the SISPROT tip. Centrifuge at 400 g for 5 min or until all liquid is passed through. Repeat this step. (4) Wash the SISPROT tip with 20 µl of wash buffer. Centrifuge at 2000 g for 0.5 min or until all liquid is passed through. (5) Infuse 2 µl of reduction buffer into the SISPROT tip by a syringe with a customized tip adapter, and incubate for 15 min at room temperature. (6) Wash the SISPROT tip with 20 µl of water. Centrifuge at 2000 g for 0.5 min or until all liquid is passed through. (7) Infuse 2 µl of digestion buffer into the SISPROT tip by a syringe with a customized tip adapter, and incubate for 60 min at room temperature (in darkness). (8) Transfer the peptides from the SCX beads to C 18 disk with 20 µl of transfer buffer. Centrifuge at 400 g for 5 min or until all liquid is passed through. (9) Wash the SISPROT tip with 20 µl of desalting buffer. Centrifuge at 2000 g for 0.5 min or until all liquid is passed through. (10) Elute the peptides with 20 µl of elution buffer or fractionate the peptides by 20 µl of fractionation buffer. Centrifuge at 300 g for 5 min or until all liquid is passed through. (11) Lyophilize the eluted peptides to dryness in the vacuum concentrator. (12) Redissolve the peptides in 10 µl of resuspension solution for LC-MS/MS analysis. Page S-4

5 Performance comparison with the capillary-based and centrifugal proteomic reactor For performance comparison among different proteomic reactors, HEK 293T cells were harvested and lysed using the modified RIPA lysis buffer 1 for the capillary-based 1 and centrifugal 2 proteomic reactor, and using the modified RCPR lysis buffer (10 mm HEPES, ph 7.4, 150 mm NaCl, 2 mm CaCl 2, 2 mm MgCl 2, 600 mm guanidine HCl, 1% DDM, and protease inhibitor mixture) for SISPROT, respectively. The cell lysates were briefly sonicated for several times and then incubated on ice for 30 min. After centrifuging at g for 15 min (4 C), the protein concentrations were measured by BCA assay (Thermo Scientific), and 6 µg proteins were processed by these three proteomic reactors without fractionation, respectively. The capillary-based proteomic reactor with column dimensions of 200 µm i.d. 4 cm was performed as described previously. 1 Briefly, the protein sample was acidified and diluted with 50 mm H 3 PO 4 in a ratio of 1:9 (v/v). Trypsin was added to the sample in a ratio of 1:5 (w/w). After sample loading, the reactor was washed with 20% (v/v) ACN in 8 mm potassium phosphate buffer (ph 3) and then washed further with deionized water. Next, the proteins were reduced using 100 mm dithiotheritol (DTT) in 10 mm ammonium bicarbonate (ph7.8) for 30 min. For alkylation and digestion, 10 mm iodoacetamide (IAA) in 100 mm Tris-HCl, ph 8 was introduced into the reactor and incubated for 2 h at room temperature. Finally, peptides were eluted using 30 µl of 200 mm ammonium bicarbonate and acidified by 3 µl of 50% FA before nano-lc-ms/ms analysis. For centrifugal proteomic reactor operation, 2 6 µg proteins, 10 µl of SCX slurry (Polymer Laboratories, Varian, Inc.), and 1.2 ml of 50% methanol, 5% FA were transferred into an 1.5 ml Eppendorf tube and mixed by vigorous vortexing for 1 min. The sample was centrifuged at 16,100 g for 2 min. After removing the supernatant, the pellet was again treated with 1.2 ml of 0.5% FA. The sample was then reduced by 20 µl of 20 mm DTT in 150 mm ammonium bicarbonate (ph 7.8) at 56 C for 15 Page S-5

6 min. Next, the sample was alkylated by 20 µl of 100 mm IAA in 150 mm ammonium bicarbonate in darkness for 15 min (at room temperature), and the reaction was quenched by the introduction of 1.2 ml of 0.5% FA containing 2 µg trypsin. After centrifugation and removing the supernatant, 20 µl of 1 M ammonium bicarbonate was added to the pellet to activate the trypsin digestion, which proceeded on a shaker at 37 C for 2 h. Finally, 300 µl of 3 M ammonium hydroxide solution (ph 12) was used to elute the peptides, which were lyophilized to dryness and then redissolved in 0.1% FA for nano-lc-ms/ms analysis. Page S-6

7 Figure S-1. Testing the binding capacity of SCX beads in the SISPROT by SDS-PAGE analysis (Coomassie brilliant blue staining) of the BSA flow through (FT). (A) The SISPROT consisted of 1 mg SCX beads and 1 plug of SCX disk (3M Empore, USA) into a 200 µl pipette tip. An aliquot of 10 µg BSA was seriatim loaded onto the SISPROT. The flow through were collected for SDS-PAGE analysis separately. (B) The SISPROT was made by packing 1 plug of SCX disk into a 200 µl pipette tip. An aliquot of 2 µg BSA was seriatim loaded onto the SISPROT. The flow through were collected for SDS-PAGE analysis separately. The results indicate that the binding capacity of 1 plug of SCX disk is 1 µg protein, while that of 1 mg SCX beads is about 30 µg proteins. Figure S-2. Common and unique proteins identified with a minimum of two peptides per protein from 20,000 HEK 293T cells in triplicate analysis. Page S-7

8 Figure S-3. Label-free quantification (LFQ) analysis of 100,000 SHED cells as processed by the SISPROT. (A-C) Correlation of the LFQ intensities of proteins between any of two replicates. (D-F) Distribution of the ratios of LFQ intensities for proteins between any of two replicates. R1, R2, and R3 represent the LFQ intensities of proteins identified from replicate 1, 2, and 3, respectively. Page S-8

9 Table S-1. Cells counted by a hemocytometer. Times Cell number Concentration * (cells/ml) Average * Concentration = (cell number)/4 (dilution ratio) Table S-2. Peptides and proteins identified from 20,000 HEK 293T cells in triplicate Replicates analysis. Identified unique peptides Identified proteins Identified proteins * Replicate 1 41,851 5,511 4,149 Replicate 2 42,543 5,642 4,265 Replicate 3 41,447 5,669 4,300 Combined results 62,241 6,552 4,890 * Proteins identified with a minimum of two peptides per protein. Table S-4. Peptides and proteins identified from 100,000 SHED cells in triplicate analysis. Replicates Identified Identified Identified unique peptides proteins proteins * Replicate 1 87,150 7,765 6,097 Replicate 2 78,211 7,257 5,633 Replicate 3 77,650 7,364 5,702 Combined results 120,456 9,078 6,924 * Proteins identified with a minimum of two peptides per protein. References (1) Ethier, M.; Hou, W.; Duewel, H. S.; Figeys, D. J. Proteome Res. 2006, 5, (2) Zhou, H.; Wang, F.; Wang, Y.; Ning, Z.; Hou, W.; Wright, T. G.; Sundaram, M.; Zhong, S.; Yao, Z.; Figeys, D. Mol. Cell. Proteomics 2011, 10, O , Page S-9