TOP-DOWN SEQUENCING IN VIBRATIONALLY COOLED MALDI FTICR MS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。自顶向下测序和蛋白质结构表征在电喷雾FTMS仪器上显示出巨大的实用性。然而，使用振动冷却（VC） MALDI FTMS仪器进行自上而下的质谱分析是完全可能的。由波士顿大学医学院NCRR质谱资源建立的振动冷却MALDI能够冷却离子的振动运动，并且是MALDI FTMS中生成完整蛋白质的关键参数。测试该技术的模型蛋白为泛素。用紫外（355 nm）和红外（2940 nm）激光从合适的基质上对完整的物种进行解吸。分子离子的SORI CAD激发产生了多个序列片段，这些片段可以形成一个蛋白质序列标签，这些序列标签足以通过数据库搜索进行鉴定。序列覆盖率约为50%。进一步的研究必须优化片段的产生和检测程序，并且必须对生物衍生（非合成）样品进行测试。更多的实验正在进行中，涉及样品制备和修改HP MALDI的离子源，以允许使用液体基质，如甘油甚至水。从液体基质中产生多电荷的完整蛋白可能会产生更好的序列覆盖率。因此，通过VC MALDI FTMS对（小）蛋白进行自上而下的测序是一种很有前途的蛋白质鉴定和结构研究技术。在振动冷却的MALDI FTMS中，首次获得了由泛素产生的自上而下的数据。这些数据已提交给同行评议的期刊发表

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. Top-down sequencing and protein structural characterization has shown great utility on electrospray FTMS instruments. It is, however, entirely possible to perform top-down mass spectrometry using vibrationally cooled (VC) MALDI FTMS instrument. The vibrationally cooled MALDI built at the NCRR Mass Spectrometry Resource at Boston University School of Medicine is capable of cooling the vibrational motion of the ions and is a critical parameter for the generation of intact proteins in MALDI FTMS. The model protein for testing this technique was ubiquitin. The intact species was desorbed from appropriate matrices using both UV (355 nm) and IR (2940 nm) laser. The SORI CAD excitation of molecular ions produced several sequence fragments from which a protein sequence tag can be formed which is more than sufficient for identification via database searching. The sequence coverage was approximately 50%. Further studies have to be done to optimize the fragment generation and detection procedure, and the method must be tested for biologically derived (non-synthetic) samples. More experiment are underway concering sample preparation and the modification of ion source of HP MALDI to allow use of liquid matrices such as glycerol or even water. Generation of multiply charged species of intact proteins from liquid matrices will likely generate improved sequence coverage. Thus, top down sequencing of (small) proteins by VC MALDI FTMS is a promising technique for protein identification and structural studies. The top down data generated from ubiquitin was obtained for the first time for vibrationally cooled MALDI FTMS. These data have been submitted for publication in a peer-reviewed journ