SUPPORT FOR A GAS-PHASE PROTEIN/PEPTIDE SEQUENCER

我们建议建立一个蛋白质微量测序设施，并 利用气相测序仪的多肽和最先进的 高效液相色谱仪。至少有八名调查人员将使用 该设施适用于各种项目：1)主要结构和 核仁非组蛋白的磷酸化和甲基化位点 将对C23、B23和160K进行分析。该设施还将用于 研究这些蛋白质与其他蛋白质相互作用的区域 大分子。2)来自其羧基末端区域的多肽 真核细胞多肽链起始因子eIF-2(α)和eIF-4A 将被测序。3)将在78 kDa上获得序列信息 Poly(A)结合蛋白和甲基化赖氨酸残基的位置将 测定小鼠红白血病细胞的伸长因子Tu。 4)家蚕氨基酰基-tRNA合成酶的一部分 将进行测序，并在亲和之后识别活动站点区域 贴标签。5)该设施将用于评估结构 小鼠单抗Mu和L链的同源性。6)至 确定每个金属辅因子是否存在单独的酶种类， 将获得有关锰和铁超氧化物歧化酶的序列数据 来自变形链球菌。7)将对鲶鱼免疫球蛋白的结构域进行测序 确定不同H链和L链的结构基础 子类。在项目1到4中获得准确的序列 信息对克隆和DNA测序项目至关重要。总而言之， 可获得的蛋白质数量有限的项目，从而创造了 高灵敏度蛋白质测序设备的必要性。

We propose to establish a facility for micro-sequencing of proteins and peptides utilizing a gas-phase sequencer and a state-of-the-art high performance liquid chromatograph. At least eight investigators will use this facility for a variety of projects: 1) The primary structures and sites of phosphorylation and methylation of nucleolar nonhistone proteins C23, B23 and 160K will be analyzed. The facility will also be used in studying regions of interaction of these proteins with other macromolecules. 2) Peptides from the carboxyl-terminal region of eukaryotic polypeptide chain initiation factors eIF-2(Alpha) and eIF-4A will be sequenced. 3) Sequence information will be obtained on a 78 kDa poly(A) binding protein and the location of methylated lysine residues will be determined on elongation factor Tu from murine erythroleukemia cells. 4) Portions of amino-acyl-tRNA synthetases from the silkworm B. mori will be sequenced and active site regions will be identified after affinity labeling. 5) The facility will be used to assess the structural homogeneity of the Mu and L chains of mouse monoclonal antibodies. 6) To determine whether separate species of enzyme exist for each metal cofactor, sequence data will be obtained on manganese and iron superoxide dismutases from S. mutans. 7) Domains from catfish immunoglobulins will be sequenced to determine the structural basis for the distinct H and L chain subclasses. In projects 1 through 4 obtaining accurate sequence information is essential for cloning and DNA sequencing projects. In all projects the amounts of obtainable protein are limited, thereby creating the necessity for a high sensitivity protein sequencing facility.