PROTEIN SEQUENCING BY TANDEM MASS SPECTROMETRY

通过串联质谱法进行蛋白质测序

• 批准号: 2178800
• 负责人: DONALD F HUNT
• 金额: $ 27.55万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-12 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178800
• 关键词: DNA binding protein; DNA directed DNA polymerase; acetylcholine; aminoacyltransferase; antigens; binding proteins; branched chain aminoacid; calcium binding protein; complementary DNA; computer assisted sequence analysis; crosslink; electrospray ionization mass spectrometry; gel electrophoresis; isoleucine; laser spectrometry; leucine; membrane channels; membrane proteins; monocyte; myosins; nicotinic receptors; oligopeptides; ovary neoplasms; phosphomonoesterases; phosphorylation; placenta; protein sequence; protein structure function; rhodopsin; vascular smooth muscle

Research to solve a number of structural problems in protein chemistry and to continue the development of instrumentation and methodology for the sequence analysis of proteins by tandem mass spectrometry is proposed. Efforts will be made; (a) to develop spectrometry for the direct sequence analysis of disease diagnostic proteins isolated at the femtomole/picomole level from cultured cells and biological fluids by two dimensional gel electrophoresis, (b) to characterize the structural modifications that proteins in the human lens undergo during cataract formation, (c) to use the laser photocrosslinking technique in combination with tandem mass spectrometry to pinpoint amino acid residues in DNA polymerase I involved in both the binding domain at which synthesis proceeds and the deoxynucleotide triphosphate binding site where the next complementary nucleotide is held, (d) to establish the primary structures of two calcium binding proteins involved in neuronal differentiation in the brain, (e) to sequence the regulatory light chain isoforms of myosin involved in maintaining high blood pressure in vascular smooth muscle, (f) to characterize the topography or folding pattern of the nicotinic acetylcholine receptor protein that makes up the ligand gated ion channel at the neuromuscular junction, (g) to locate the nine phosphorylated residues in the visual protein, rhodopsin, and to determine the order in which these residues are phosphorylated as a first step in understanding the role that phosphorylation plays in regulation of the visual process at the molecular level, (h) to establish the primary structures of human monocyte chemoattractant proteins involved in modulating the immune response to antigens, and (i) to sequence aminoacylase and two phosphotyrosyl phosphatases involved in cell proliferation. Efforts will also be made to reduce the amount of sample required for analysis on the newly acquired Model 270 triple quadrupole mass spectrometer to the low picomole range and to extend the usable mass range of this instrument beyond m/z 3,000. Capillary electrophoresis will be interfaced to the triple quadrupole and quadrupole FTMS instruments via electrospray ion sources to facilitate online analysis of protein and oligopeptide samples separated by this extremely powerful technique. Fragmentation of multiply charged peptide ions will be studied and conditions that allow differentiation of leucine and isoleucine residues by the process of collision activated dissociation or surface induced dissociation will be explored.

研究解决蛋白质化学中的一些结构问题， 继续发展监测手段和方法， 提出了通过串联质谱法进行蛋白质序列分析。 将作出努力;（a）发展直接序列的光谱测定法 以飞摩尔/皮摩尔分离的疾病诊断蛋白的分析 从培养的细胞和生物液体中通过二维凝胶水平 电泳，（B）表征结构修饰， 白内障形成过程中人类透镜中的蛋白质，（c）使用 激光光交联技术与串联质谱联用 光谱法来精确定位DNA聚合酶I中的氨基酸残基 在合成进行的结合结构域和 脱氧核苷酸三磷酸结合位点， （d）建立两个钙的一级结构 参与脑中神经元分化的结合蛋白，（e） 序列的调节轻链亚型的肌球蛋白参与 维持血管平滑肌中的高血压，（f） 表征烟碱的拓扑结构或折叠模式 构成配体门控离子通道的乙酰胆碱受体蛋白 在神经肌肉接头处，（g）定位九个磷酸化的 残基的视觉蛋白，视紫红质，并确定顺序， 这些残基被磷酸化作为理解的第一步， 磷酸化在调节视觉过程中的作用， （h）建立人的一级结构 参与调节免疫的单核细胞趋化蛋白 对抗原的应答，和（i）对氨基酸酰化酶的序列和两个 参与细胞增殖的磷酸酪氨酸磷酸酶。 努力将 也可以减少分析所需的样品量， 新获得的270型三重四极杆质谱仪低 皮摩尔范围，并扩展该仪器的可用质量范围 超过m/z 3000 毛细管电泳将与 通过电喷雾离子的三重四极杆和四极杆FTMS仪器 促进蛋白质和寡肽样品在线分析的来源 被这种极其强大的技术分开。 乘法分裂 带电肽离子将被研究， 亮氨酸和异亮氨酸残基的区分， 碰撞活化解离或表面诱导解离将是 探讨了