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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 振动冷却MALDI-FTMS的改进和优化工作仍在继续。今年，我们对与弗劳恩霍夫研究所合作设计和建造的XY舞台进行了进一步的测试和评估。这一阶段是MALDI-FTMS完全自动化的关键组成部分。 这一阶段的设计参数被定义为处理典型的蛋白质组型样本。许多蛋白质组学实验使用的机器人是为处理约76×毫米大小的微滴定板而设计的。因此，要求舞台的全程行程至少为100x100毫米。对于所使用的MALDI傅立叶变换质谱计，点到点的旅行时间不是实验速度的限制因素，因此2 mm/s的旅行被定义为允许工作台在0.5秒内定位到1 mm外的下一个点。定位精度被定义为1um，这比标准MALDI实验所需的精度高得多，在标准MALDI实验中，激光光斑的直径通常为50um，但将允许在MALDI成像模式下使用工作台。除此之外，工作台必须是真正的真空兼容的，必须垂直安装，并且不会产生可检测到的射频干扰噪声，并且必须占用最小的空间，以便真空室可以尽可能地小以提高抽气性能。 二维工作台是单块设计，将两轴电机、直线编码器和位置开关全部集成到中央块中。该系统使用滚珠丝杠和十字滚子轴承，在真空中只需要最少的润滑；只需要很少的润滑就可以由低蒸汽压扩散泵油提供。为了将电机安装到这个滑块上，它们安装在铜支架上(将它们的热量输送到充当散热器的滑轮上)，并使用滑轮将它们的扭矩传递到滚珠丝杠上。这些皮带轮的皮带实际上是小的电焊不锈钢带，为了使皮带轮保持在皮带轮的中心，皮带轮被磨得略微凹陷。拉紧固定螺丝安装在滑轮块中，以便在测试过程中正确地拧紧滑轮。为了防止过度确定滚珠丝杠的位置，滚珠丝杠只安装在两个地方：滑轮轴承总成和托板。 舞台本身被设计成三个部分，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心模块上(并散热)。直线编码器滑板安装在上板和下板上。这三块都是黑色的阳极氧化，以提高表面硬度，但更重要的是，改善块的辐射冷却。所有导线也都包含在块中。电机和电子设备将它们的热负荷倾倒到中心块中，中央块只能通过轴承的传导冷却和辐射冷却来散热。传导冷却效率非常低，因为轴承是不锈钢的(热传递能力非常低)，接触面积非常小。人们曾考虑使用铜带或编织带来提高导热效率，但通过快速计算铜带与辐射冷却速率的比较，发现通过铜带的换热可以忽略不计。有几个用于交叉滚子轴承的定位螺钉、钢丝轨迹和用于安装电机、轴承和电子设备的螺栓孔。所有这些孔都有通风口，以最大限度地减少虚拟泄漏。 该系统使用四个位置编码器、两个用于确定位置的直线编码器和两个安装在电机背面的旋转编码器来确定速度。该系统使用光学限位开关。开关是安装在中央台体上的光电二极管/光电晶体管对，该对被安装在上下台板上的铝旗打断。每个轴安装两个这样的开关。当工作台移动到其运动范围的末尾时，该标志阻止光电二极管向光电晶体管发送信号，并且电子设备向工作台发出停止的信号。这种系统虽然类似于摇杆开关或磁开关，但涉及的部件更少，定位精度更高。 样品平移平台需要大量的真空电馈线(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，由于它安装在对射频干扰噪声高度敏感的FTMS上，因此馈线和电缆必须得到良好的屏蔽。 此样品平移工作台的真空室被设计为尽可能小(为了有效的下压)，但足够大，以允许工作台的全部运动范围。示例翻译阶段控件是作为一组命令开发的，可以从任何基于Microsoft Windows的编程语言的动态链接库(DLL)中调用这些命令。这里使用的语言是在Microsoft Visual C++版本6中实现的C++。现在已经构建了舞台的副本，用于在BUSM心血管蛋白质组学中心组装的低温FTM。

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 we carried out further testing an evaluation of the XY stage that was designed and constructed in collaboration with the Fraunhofer Institute. This stage is a 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 um, which is much more accurate than is needed for the standard MALDI experiment in which the laser beam spot is often 50 um diameter, but will allow the stage to be used in a MALDI imaging mode. 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 minimal 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 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 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 its range of motion, this flag blocks the photodiode's signal to the phototransistor, and the electronics 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 an FTMS 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. A copy of the stage has now been constructed for use with the cryogenic FTMS being assembled within the BUSM Cardiovascular Proteomics Center.