ION-SPRAY TRIPLE QUADRUPOLE PROTEIN MASS SPECTROMETER

请拨经费购买一台离子喷雾三重四极质谱计 在涉及蛋白质的生物研究中的使用。该仪器请求一个 溶液中的各种蛋白质样品，总量从几个到一个 每个实验几十个皮摩尔(约1微克；20微微克 25 kDa蛋白质为0.5微克)。生成的信息包括精确的 完整蛋白质或蛋白质混合物组分的质量(质量 25 kDa蛋白质的精确度约为+/-3道尔顿)。单独的多肽， 即使在化学或酶消化中存在的复杂混合物中 一种蛋白质的几十个皮摩尔，可以直接研究，也可以事后研究 用反相高效液相色谱法预先分离多肽。对于最高达 20或30个氨基酸的长度，质量精度比1道尔顿更好 获得。这种精确的质量信息是一种通用的检测方法 以及定义翻译后修饰，包括二硫键 蛋白质的位置。这些数据还可以作为一种精确的方式 将基因与其翻译产物相关联，不仅揭示了 共价修饰，以及蛋白质的N-端或C-端的“剪裁”。 所描述的仪器的另一项功能是质谱学- 质谱学实验，使用胶原蛋白诱导解离 从混合物的单个组分生成定义序列的片段 多肽或蛋白质的。通常情况下，这样的质谱图太复杂了 完整的解释，但这些数据在定义 翻译后修改的确切性质和地点，例如 多肽磷酸化的确切位置和性质。碳水化合物部分 对糖肽的影响也可以产生结构定义的碎片模式。 更罕见的是，MS/MS可以从以下来源生成有限的氨基酸序列数据 被阻断的蛋白质，特别是来自胰酶消化的蛋白质，它可以提供 可解释的碎片模式多达20个：大约存在的多肽 在摘要中。这在生成寡核苷酸时很有用 分离出相应的基因。所描述的功能具有 在广泛的生物学研究中的重要用途。这台仪器将 对NIH赞助的一些项目的效果产生了深远的影响 程序。

Funds are requested for a ionspray triple-quadrupole mass spectrometer for use in biological research involving proteins. The instrument requested a wide range of protein samples in solution, in total amounts of a few to a few tens of picomoles per experiment (approx. 1mugram; 20 picomoles of a 25kDa protein is 0.5mugram). Information generated includes the precise mass of the intact protein or components of a protein mixture (mass accuracy; approx.+/-3dalton for a 25kDa protein). Individual peptides, even in the complex mixtures present in the chemical or enzymatic digest of a few tens of picomoles of a protein, can be studies directly or after prior separation of the peptides by reverse phase HPLC. For peptides up to 20 or 30 amino acids in length, mass accuracy of better that 1 dalton is obtained. Such precise mass information is a general method for detecting and defining post-translational modifications, inc. disulfide bond locations, of proteins. The data also serve as a precise way of correlating a gene with its translation product, revealing not only covalent modifications but also N- or C-terminal "clipping" of the protein. An additional capability of the instrument described is mass spectrometry- mass spectrometry experiments, using Collison-Induced-Dissociation to generate sequence-defining fragments from a single component of a mixture of peptides or proteins. Frequently, such mass spectra are too complex for complete interpretation, but the data can be enormously useful in defining the exact nature and site of post-translational modifications, such as the exact site and nature of phosphorylation of peptide. Carbohydrate moieties on glycopeptides can also yield structure-defining fragmentation patterns. More rarely, MS/MS can generate limited amino acid sequence data from blocked proteins, especially from tryptic digests which can give interpretable fragmentation patterns in up to 20: or so of peptides present in a digest. This can be useful in generating oligonucleotides for isolation f the corresponding gene. The capabilities described have important uses in a wide range of biological research. The instrument will have a profound impact on the effectiveness of a number of NIH-sponsored programs.