VIBRATIONALLY COOLED MATRIX-ASSIST LASER DESORPTION/IONIZATION FT MASS SPECTROM

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。振动冷却MALDI-FTMS的改进和优化仍在继续。今年，我们与Fraunhofer合作建造的XY载物台进行了安装和测试。该阶段是MALDI-FTMS完全自动化的第一个关键组成部分。 定义该阶段的设计参数以处理典型的蛋白质组类型样本。许多蛋白质组学实验使用的机器人被设计为与尺寸为~76 x 89 mm的微量滴定板一起工作。因此，要求载物台具有至少100 x 100 mm的全行程。对于所使用的MALDI傅里叶变换质谱仪，从点到点的行程时间不是实验速度的限制因素，因此定义2 mm/sec的行程，因为它允许载物台在0.5秒内定位到下一个1 mm远的点。定位精度被定义为1？m，这是更准确的比所需的标准MALDI实验中，激光束斑往往是50？m直径，但允许在未来的MALDI成像模式下使用的阶段。除此之外，工作台必须是真正的真空兼容，必须垂直安装，并且不产生可检测的RF干扰噪声，并且必须占用尽可能小的空间，以便真空室可以尽可能小，以提高泵送性能。 二维平台是一个单一的块设计与两个轴电机，线性编码器和位置开关都集成到中央块。该系统使用滚珠丝杠和交叉滚子轴承，这将需要在真空中进行最少的润滑;所需的少量润滑由低蒸汽压扩散泵油提供。为了将电机安装到这个块中，它们安装在铜支架上（将它们的热量倾倒到块中？其用作散热器），并且滑轮用于将它们的扭矩传递到滚珠丝杠。 这些滑轮的皮带实际上是小型电焊不锈钢带，为了保持它们在滑轮上居中，滑轮被磨得稍微凹进去。将张紧固定螺钉安装到测试块中，以便在测试期间正确拧紧滑轮。为了防止过度确定滚珠丝杠的位置，它只安装在两个地方：滑轮轴承组件和板。 舞台本身设计成三块，一块上下板和中央块。电机、限位开关和线性编码器电子设备都安装在中央块上（并散热）。线性编码器滑块安装在上下板上。这三件是黑色阳极氧化，以提高表面硬度，但更重要的是，以提高辐射冷却的块。所有导线布线也包含在块中。电机和电子设备将其热负荷倾倒到中央块中，中央块只能通过轴承处的传导冷却和辐射冷却来散热。传导冷却是非常低效的，因为轴承是不锈钢的（非常低的传热能力），接触面积非常小。铜带或编织被认为是为了提高传导热传递率，但快速计算这种带的热传递与辐射冷却率相比（假设黑体？因此进行了注释）表明通过铜带的热传递可以忽略不计。有几个定位螺钉用于交叉滚柱轴承、导线迹线和用于安装电机、轴承和电子设备的螺栓孔。所有这些孔都是通风的，以尽量减少虚拟泄漏。 该系统使用四个位置编码器，两个线性编码器用于确定位置，两个旋转编码器安装在电机背面用于确定速度。该系统使用光学限位开关。该开关基本上是一个光电二极管/光电晶体管对安装在中央台体，这是中断的铝旗安装在上，下阶段板。每个轴安装两个这样的开关。当舞台走到尽头的时候？的运动范围，这个标志将阻止光电二极管？的信号，光电晶体管，电子将信号阶段停止。这种系统虽然类似于摇臂开关或磁性开关，但涉及更少的部件和更高的定位精度。 样品平移台需要大量的真空电馈电器（46）来控制2个电机、4个编码器、4个限位开关和一个热电偶。此外，因为这是安装在傅立叶变换离子回旋共振质谱仪，具有高灵敏度的RF干扰噪声，这是至关重要的，馈通和电缆被很好地屏蔽。 用于该样品平移台的真空室被设计为尽可能小（用于有效抽气），但足够大以允许台的全范围运动。示例翻译阶段控件是作为一组命令开发的，可以在任何Microsoft窗口中从动态链接库（dll）调用。的编程语言。这里使用的语言是在Microsoft Visual C++版本6中实现的C++。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The improvement and optimization of the Vibrationally Cooled MALDI-FTMS continues. This year saw installation and testing of the XY stage that we have been building in collaboration with Fraunhofer. This stage is the first critical component for full automation of the MALDI-FTMS. The design parameters for this stage were defined to handle the typical proteomic type sample. Many proteomic experiments use robots that are designed to work with microtiter plates which are ~76 x 89 mm in size. Therefore, the stage was required to have full travel of at least 100 x 100 mm. For the MALDI Fourier transform mass spectrometer used, the travel time from spot to spot is not the limiting factor in the experiment speed, so a travel of 2 mm/sec was defined as it would allow the stage to position to the next spot 1 mm away in 0.5 seconds. The positioning accuracy was defined to be 1 ?m, which is much more accurate than is needed for the standard MALDI experiment in which the laser beam spot is often 50 ?m diameter, but allows the stage to be used in a MALDI imaging mode in the future. Aside from this, the stage had to be truly vacuum compatible, had to be mounted vertically, and to generate no detectable RF interference noise, and had to take up the minimal possible space so that the vacuum chamber could be made as small as possible for improved pumping performance. The two-dimensional stage is a single block design with the two axis motors, linear encoders, and position switches all integrated into the central block. The system uses ballscrews and crossed roller bearings which will require minimal lubrication in vacuum; what little lubrication is needed is provided by low vapor pressure diffusion pump oil. In order to build the motors into this block, they are mounted on copper brackets (to dump their heat into the block ? which acts as a heat sink) and pulleys are used to transfer their torque to the ballscrew. The belts for these pulleys are actually small electrowelded stainless steel bands, and in order to keep them centered on the pulleys, the pulley wheels are ground to be slightly concave. Tensioning set screws are mounted into the block to allow the pulleys to be tightened properly during testing. In order to prevent over determination of the position of the ballscrew, it is mounted in two places only: the pulley bearing assembly and the plate. The stage itself is designed in three pieces, an upper and lower plate, and the central block. The motors, limit switches, and linear encoder electronics are all mounted (and heat sinked) to the central block. The linear encoder slides are mounted to the upper and lower plates. The three pieces are black anodized to improve surface hardness, but even more importantly, to improve radiative cooling of the blocks. All wire routes are also contained within the block. The motors and electronics dump their heat loads into the central block, which is only able to dissipate that heat through conductive cooling at the bearings and radiative cooling. The conductive cooling is very inefficient since the bearings are stainless steel (very low heat transfer capacity) and the contact area is very low. A copper band or braid was considered for improving the conductive heat transfer rate, but a quick calculation of the heat transfer of such a band compared to the radiative cooling rate (assuming black body ? hence the annodization) showed that the heat transfer through the copper band was negligible. There are several positioning screws for the crossed roller bearings, wire traces, and bolt holes for mounting the motors, bearings, and electronics. All of these holes are vented to minimize virtual leaks. The system uses four position encoders, two linear encoders for determining position, and two rotary encoders mounted on the back of the motors for determining velocity. The system uses optical limit switches. The switch is basically a photodiode/phototransistor pair mounted on the central stage body which is interrupted by an aluminum flag mounted on the upper and lower stage plates. Two such switches are mounted for each axis. When the stage moves to the end of it?s range of motion, this flag will block the photodiode?s signal to the phototransistor, and the electronics will signal the stage to stop. Such a system, while similar to a rocker switch or magnetic switch involves fewer parts and greater positioning accuracy. The sample translation stage requires a large number of vacuum electrical feedthroughs (46) to control 2 motors, 4 encoders, 4 limit switches, and a thermocouple. Furthermore, because this is being mounted on a Fourier transform ion cyclotron resonance mass spectrometer which has high sensitivity to RF interference noise, it was critical that the feedthrough and cables be well shielded. The vacuum chamber for this sample translation stage was designed to be as small as possible (for efficient pumpdown) but large enough to allow the full range of motion for the stage. The sample translation stage control was developed as a set of commands that can be called from a dynamically linked library (dll) in any Microsoft window?s based programming language. The language used here was C++ as implemented in Microsoft Visual C++, version 6.