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Research paper : A systematic analysis of protein interaction networks leading to the drug discovery (S. Iemura et al.)−116 Synthesiology - English edition Vol.1 No.2 (2008) automated high flow-rate mixing module.Describing this idea in one sentence, it is “mixture is made homogenously in a larger world, and then delivered to a smaller world,” much like “Columbus’ egg.” Although it was important to make sure that the solvents in each step would not mix with each other, we took advantage of the disadvantage of a dual-system pump where the solvents in a microflow channel do not mix (lower part of Figure 2). This method enabled extremely high reproducibility as well as continuous operation and automation of LC. Using this method, HPLC at lower than 100 nL/min with direct flow without split was installed online with mass spectrometer for the first time in the world[1]. As a result, a high sensitivity of 20~50 times the conventional method was achieved.We also improved the analytical environment to maximize the high sensitive analysis. Even if we achieved sensitivity that allowed analysis of microquantity samples, since massive amounts of human keratin existed in general experimental environment, the signal of microquantity sample would be overpowered by contamination of air-borne keratins. Also, since the analytical channel consisted of a thin capillary with internal diameter of 10 micrometer to eliminate dead volume as much as possible, the channel became clogged readily by dust particles in the atmosphere. Therefore, continuous operation in a general environment where people walk in and out was impossible. Although elimination of dust particles and keratin were not easy, we started to use the prototype system for routine anayses.In 2000, we started analysis using actual samples at Tokyo Metropolitan University where the technological development was carried out. Since there was no clean room at that time, we evaded the issue of experimental environment by limiting entry and exit of people to the analysis lab. The lab was thoroughly organized to eliminate dust source and covered with antistatic sheet as much as possible to prevent adsorption of dust by static electricity. Entry to the room wearing raised fabric clothes was prohibited. Also, after opening and closing the door of the lab, we waited until the dust settled, and only after then did we start the analysis.The excitement we felt when over 100 proteins were identified at once for first time using this system was unforgettable. We gradually obtained cooperation of people who appreciated the (44)−Fig. 2 Comparison of new and conventional technologies.In conventional technology, elution gradient is created by a dual-system pump. Concentration gradient is created by shifting the transfer rate of pump A that delivers initial solvent and pump B that delivers elution solvent. However in this method, low speed mixing is not possible since the dead volume is large. Therefore, low speed is achieved by installing a splitter between the analytical columns and by discarding most of the liquid. The figure shows that the flow rate is decreased from 5000 nL to 500 nL/min by discarding 1/10 of the liquid (upper part).In the new technology, reservoirs with multiple branches are filled with elution solvents in steps using a separate pump system. Elution gradient is created by rotating the port valve and pushing out each step with a single-system low-speed pump. There is no dead volume and it does not require a splitter (lower part).In conventional technology, sample preparation by mass culture was necessary for a single analysis. It was common to conduct 100 L culture using jar fermenter. Several months were required to prepare for an analysis. Using the technology we developed, we achieved sensitivity where several analyses would be possible with samples from 10 mL culture. In this scale, only a few days are necessary for sample preparation, and several different samples can be prepared simultaneously.5000 nL/min500 nL/min100 nL/minMSSeveral monthsLow-speed liquid chromatography using split (conventional technology)Jar fermenter (100 L culture)Waste liquidMass spectrometerRP columnA few daysMSMass spectrometerNew technology in which minute concentration gradient is createdRP column10 mL cultureElution gradient

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