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Interface Solution Isoelectric Focusing with in situ MALDI-TOF Mass Spectrometry Shujun Wang,1, 2 Suming Chen,3 Jianing Wang,3 Peng Xu,2 Yuanming Luo,2 Zongxiu Nie,3,* Wenbin Du2, 1* Supporting Information Chemicals and Materials: All water used was deionized water from a Hitech Master-S UVF Water Purification System (Shanghai, China). Ribonuclease A from Bovine Pancreas (RNase A, MW: 13.7 kDa, pI=7.8) was obtained from Sangon Biotech. (Shanghai, China). Green Fluorescent Protein (GFP, MW: ~29 kDa, pI=4.7~4.9) was obtained from Sino Biological Inc. (Beijing, China). Bovine Serum Albumin (BSA, MW: ~66 kDa, pI=5.6), Fluorescein isothiocyanate (FITC), Sinapic acid (SA), and 1H, 1H, 2H, 2H-Perfluoro-1-octanol (PFO) were obtained from Sigma Aldrich. Pharmalyte (pH 3-10) was purchased from GE Healthcare (Sweden). Methylcellulose and Tetramethylethylenediamine (TEMED) were purchased from Yuanju Biotech. (Shanghai, China). α-cyano-4-hydroxycinnamic acid (CHCA) was purchased from TCI Company (Shanghai, China). Tridecafluoro-1,1,2,2-tetrahydrooctyl-1- trichlorosilane was obtained from Fluorochem Ltd. (Derbyshire, UK). FC-40 oil was obtained from 3M (St. Paul, MN). Soda-lime glass (0.7 mm thick) with chromium and AZ1500 photoresist coating were purchased from Telic Company (Valencia, CA). Amorphous diamond coated drill bits were obtained from Harvey Tool (Rowley, MA). Teflon tubing (O.D. 150 μm, I. D. 100 μm) was purchased from Zeus (Orangeburg, SC). Conductive tape was obtained from Kaivo Optoelectronic Technology Co. (Zhuhai, China). Instruments and Computer Programs: Pipettes were obtained from Eppendorf (Hamburg, Germany). Stereoscope was purchased from Shunyu Optical Technology (Ningbo, China). A 1 custom made DC power supply was used for IEF. A transilluminator with an excitation wavelength of 470 nm and an emission wavelength of 510 nm was purchased from Zeesan Biotechnology (Xiamen, China). All fluorescence images were taken on a Nikon Eclipse-Ti inverted fluorescence microscope (Tokyo, Japan). Images were analyzed with ImageJ software. MALDI-TOF MS analysis of samples was carried out on a Bruker ultrafleXtreme instrument (Bruker Daltonics, USA) equipped with a 355 nm Nd-YAG laser, ion acceleration voltage of 25 kv and pulse extraction delay time of 283 ns. Each spectrum obtained was the summation of 500 laser shots to maximize the signal to noise ratio. MS results were analyzed with Bruker Daltonics FlexAnalysis 4.0 Software and MALDI Imaging MS was conducted and analyzed with Bruker Daltonic fleximaging 3.0 software. FITC-labeling procedure for proteins: FITC labeling was carried out according to the product instructions. Briefly, proteins were dissolved in 40 mmol/L carbonate buffer to concentration of 10 mg/mL. FITC solution (1.5 mg/mL) was prepared in 40 mmol/L carbonate buffer. Then FITC solution was slowly added into the protein solution in the dark with stirring. After that, the mixture was incubated in the dark with stirring at 4 ºC for 12 hours. Finally, the reaction product was purified with a dialysis bag (MWCO: 4000 Da) in 1X PBS for 24 hours to remove the excess FITC and other substances with low molecular weight. Depositing of matrix: Matrix for MALDI-TOF MS analysis were prepared by dissolving matrix in 50% acetonitrile, 49.9% D. I. water, and 0.1% trifluoroacetic acid. MALDI matrix solutions for protein samples and the CSF sample were 10 mg/mL sinapic acid (SA) and 5 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) respectively. Commercial-available Matrix Sprayers can be used for applying matrix on the glass plate automatically. In this work, 10-nL matrix solution was manually deposited into each microwell for co-crystallization with the sample. A 50 µL Hamilton glass syringe was placed on a Harvard Apparatus syringe pump (Holliston, MA). The syringe was connected with a piece of Teflon tubing (150 μm O. D., 100 μm I. D., 10 cm long) and filled with matrix solution. The tip of the 2 tubing was placed in the microwells under stereoscope, and matrix solution was injected into each microwell with a preset target volume of 10 nL at a flow rate of 0.3 μL/min. 3 Figure S1. The slipping and freezing procedure prior to MS detection. a-e) Cross-section schematics of the device operation after sIEF prior to MS; f-g) Photographs of the microwell array containing blue dye solution slipped to the etched open area with and without the freezing step. 4 Figure S2. Steps for generate identical IEF segments on the two glass plates. The device (a) was slipped horizontally to overlap the microwell array on the top plate and bottom plate (b), and slipped again to divide the mixed microwells and generate duplicates (c). 5 Figure S3. Photograph of the glass plates with conductive copper tapes on the MALDI target plate. White cross on the plate are markers for alignment. 6 Figure S4. Mass spectra of CSF sample indicating that there are no metabolites with molecular weight larger than 600. The CSF sample were prepared same as sIEF experiments (added with carrier ampholyte, TEMED and glycerol, and cocrystallized with CHCA). The data are overlay of 14 Mass spectra each was the summation of 500 laser shots to enhance to maximize the signal to noise ratio. 7 Table S1. Peaks observed in mass spectra with mass-to-charge ratio (m/z) from 100 to 600. No. (1) control solution of carrier ampholyte (m/z) (2) directly deposited CSF sample with carrier ampholyte (m/z) 1. 2. 3. 100.904 102.828 114.932 100.845 102.787 114.893 4. 116.884 116.880 5. (3) CSF sample separated by sIEF on chip (m/z) 100.9200 pI -- 114.6197 -- 116.4879 -- 123.4551) -- 130.8108 -- 6. 130.967 130.920 7. 144.719 144.967 8. 146712 146.973 147.0020 -- 9. 150.172 150.573 150.1157 -- 10. 159.053 159.135 158.8340 -- 11. 172.774 173.059 172.5342 -- 12. 176.174 -- 13. 176.2706 -- 186.85712) 5.09 14. 190.838 190.5935 -- 15. 213.064 213.0120 -- 16. 234.80783) -- 17. 236.13004) -- 18. 264.07645) -- 19. 275.28566) -- 20. 295.21327) -- 21. 296.50908) -- 22. 303.30889) -- 23. 319.499910) -- 24. 336.313811) 25. 337.777012) -- 26. 347.523013) -- 27. 372.692 172.84 372.4325 28. 374.013014) -- 29. 378.659015) 4.9 30. 380.528116) -- 31. 382.104017) -- 32. 403.569318) -- 33. 406.879 83.7 406.6830 34. 408.197019) -- 35. 420.657020) -- 36. 442.801721) -- 37. 475.183922) -- 38. (4) peak heights ratio of column (3) over column (2)a) 481.029 65.87 480.7886 39. 503.829823) 4.6 40. 515.661824) -- 41. 556.762425) -- 42. 559.876026) -- 8 1)-26) a) 26 new Peaks were discovered after the sIEF-MALDI SlipChip; Those peaks observed in the control solution, and peaks observed exclusively on sIEF SlipChip were not calculated. 9 Table S2. Prediction of possible metabolites in the CSF microdialysate sample based on the peaks obtained by MALDI imaging (in positive ion mode). m/z of peaks Possible metabolites Average molecular weight HMDB ID a) a 116.699 L-Proline 115.1300 HMDB00162 147.404 Acetylcholine 146.2074 HMDB00895 L-Lysine 146.1876 HMDB00182 148.023 L-Glutamic acid 147.13 HMDB00148 174.950 L-Arginine 174.201 HMDB00517 176.394 N-Acetylaspartic acid (NAA) 175.14 HMDB00812 186.865 Phosphoserine 185.0725 HMDB00272 189.141 Homo-L-arginine 188.2275 HMDB00670 N6,N6,N6-Trimethyl-L-lysine 188.2673 HMDB01325 337.759 Kyotorphin 337.3742 HMDB05768 556.762 Enkephalin L 555.6227 HMDB01045 The Human Metabolome Database (HMDB), http://www.hmdb.ca/ 10