Introduction 1 3 4 6 7 8 9 10 12 13 17 18 19 20 13 21 22 23 24 25 26 27 Experimental Materials The SPR equipment used was from IBIS Technologies (currently available from Eco Chemie, Utrecht, The Netherlands) equipped with a 200-μL polycarbonate cuvet. The gold-sensor disks, purchased from SSENS (Hengelo, The Netherlands), were positioned on the IBIS-prism using index-matching oil from R.P. Cargille Laboratories (Cedar Grove, USA). PEEK nuts, unions, tubing, and loops were from Upchurch (Santa Monica, USA). Manual injections during the preparation of the reactors were performed using a Rheodyne 7010 injector (Inacom, Veenendaal, The Netherlands) equipped with a 1-mL PEEK loop. Model 10ADvp HPLC pumps from Shimadzu (Kyoto, Japan) were used for reactor preparation and activity determinations. The water used for washing and to prepare buffers was produced by a Sartorius Arium 611 ultrapure water system (Nieuwegein, The Netherlands; conductivity >18.2 MΩ cm). The model ABS759A UV absorbance detector was equipped with a capillary flow-cell (75-μm i.d.) and was obtained from Applied Biosystems (Nieuwerkerk a/d IJssel, The Netherlands). 18 18 2 N N N 28 −1 −1 −1 2 −1 Methods 1 Fig. 1 a b c 2 b  −1 −1 −1 −1 −1 SPR experiments −1 −1 −1 1 −1 −1 −1 −1 1 −1 −1 Preparation of dextran-coated fused-silica capillaries −1 −1 −1 −1 −1 −1 1 Enzyme activity determination −1 −1 −1 −1 −1 2 On-line protein digestion in micro-HPLC 2 Fig. 2 Set-up used for on-line protein digestion using a trypsin-modified fused-silica capillary. For detection a PDA or an ion-trap MS was used  −1 http://www.matrixscience.com Results and discussion Surface chemistry and enzyme immobilization 29 n –2 −2 n −2 n 23 30 32 30 −2 Characterization of the enzyme reactor 3 Fig. 3 a b circles triangles b −1 −1 −1 −1 −1  3 −1 −1 −1 −1 −1 −1 33 34 34 The stability during operation of the open-tubular reactor was tested at 37 °C and pH 8.5. Activity tests using the model substrate show that the activity of a reactor that is continuously in operation, is constant for at least two weeks. The enzyme trypsin dissolved in digestion buffer and incubated for 24 h at 37 °C loses 60% of its activity, and after three days no activity is measured. On-line digestion of proteins 1 Table 1 n  capillary ID (μm) reactor volume (μL) amount injected digestion time (s) % undigested protein (SD) −1 (pmol) 75 1 248 20 60 65.9 (4.8) 75 1 124 10 60 35.8 (3.4 75 1 12.4 10 60 23.6 (1.3) 50 1 248 20 60 14.0 (1.9) 50 1 124 10 60 2.3 (2.0) 50 2.75 248 20 165 0 50 2.75 124 10 165 0 50 2.75 12.4 10 165 0 n 1 −1 4 4 4 −1 −1 4 −1 4 4 Fig. 4 a −1 b −1 c b −1  2 −1 3 −1 5 5 Table 2 n -1 digestion time (s) sequence coverage (%) total no peptides matched Mascot score undigested protein (%) 10 17 66 11 347 60.3 5 33 66 21 501 31.8 2 83 66 23 649 18.5 1 165 87 27 833 0 Table 3 −1 Peptide sequence m z m z Position Missed cleavage Cytochrome C (sequence coverage 87%) 1....IFVQKCAQCHTVEK 1632.8 816.9 (2+) 9–22 1 2....HKTGPNLHGLFGR 1432.8 717.1 (2+) 26–38 1 3....TGPNLHGLFGR 1167.6 584.6 (2+) 28–38 0 4....TGPNLHGLFGRK 1295.7 648.6 (2+) 28–39 1 5....KTGQAPGFTYTDANK 1597.8 533.4 (3+) 39–53 1 6....TGQAPGFTYTDANK 1469.7 735.6 (2+) 40–53 0 7....TGQAPGFTYTDANKNK 1711.8 571.4 (3+) 40–55 1 8....GITWKEETLMEYLENPKK 2208.1 736.8 (3+) 56–73 2 9....EETLMEYLENPK 1494.7 748.0 (2+) 61–72 0 10  EETLMEYLENPKK 1622.8 812.1 (2+) 61–73 1 11...YIPGTK 678.4 678.1 (1+) 74–79 0 12...MIFAGIKK 906.5 454.0 (2+) 80–87 1 13...KTEREDLIAYLKK 1477.8 739.6 (2+) 88–99 2 14...TEREDLIAYLKK 1349.7 675.5 (2+) 89–99 1 15...EDLIAYLK 963.5 482.6 (2+) 92–99 0 16...EDLIAYLKK 1091.6 546.6 (2+) 92–100 1 Myoglobin (sequence coverage 88%) 1....GLSDGEWQQVLNVWGK 1814.9 908.7 (2+) 1–16 0 2....VEADIAGHGQEVLIR 1605.8 803.6 (2+) 17–31 0 3....VEADIAGHGQEVLIR 1605.8 536.1 (3+) 17–31 0 4....LFTGHPETLEK 1270.7 636.0 (2+) 32–42 0 5....HLKTEAEMK 1085.6 543.5 (2+) 48–56 1 6....HGTVVLTALGGILK 1377.8 689.7 (2+) 64–77 0 7....HGTVVLTALGGILKK 1505.9 502.7 (3+) 64–78 1 8....KKGHHEAELKPLAQSHATK 2109.1 703.7 (3+) 78–96 2 9....KGHHEAELKPLAQSHATK 1981.0 661.1 (3+) 79–96 1 10  GHHEAELKPLAQSHATK 1853.0 618.8 (3+) 80–96 0 11...YLEFISDAIIHVLHSK 1884.0 628.8 (3+) 103–118 0 12...HPGNFGADAQGAMTK 1500.7 751.5 (2+) 119–133 0 13...ALELFRNDIAAK 1359.8 680.6 (2+) 134–145 1 14...YKELGFQG 940.5 471.0 (2+) 146–153 1 Fig. 5 −1 a 3 b m z  15 35 −1 −1 3 6 35 Fig. 6 −1 a 3 b m z  30 36 29 37 38 13 14 39 41 30 34 11 15 35 36 42 43 12 Conclusions To study the immobilization of the protein trypsin in a fused-silica capillary, a number of surface modifications was tested. In order to investigate the amount of protein that can be covalently attached to these surfaces, SPR sensor disks are modified with these coatings to mimic the capillary surface. The SPR measurements show that the best results were obtained using a dextran coating with an intermediate layer. The resulting open-tubular trypsin reactors having a pH optimum of pH 8.5 display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. −1 The open-tubular reactor can be produced easily, reproducibly, and inexpensively, and can be used for other applications such as enzyme-inhibitor studies. Future research will focus on the development of miniaturized multi-dimensional analysis systems employing on-line digestion using these IMERs. The capillary enzyme reactors show no backpressure and seem promising for coupling to other analytical techniques such as capillary electrophoresis and surface plasmon resonance.