CHARACTERIZATION AND LABELING EFFICIENCY OF 13C1-ALA

13C1-ALA 的表征和标记效率

• 批准号: 7956037
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956037
• 关键词: Acetonitriles; Algorithms; Blood capillaries; Buffers; Computer Retrieval of Information on Scientific Projects Database; Data; Databases; Digestion; Dithiothreitol; Enzymes; Formic Acids; Fourier Transform; Funding; Grant; Institution; Iodoacetamide; Ions; Label; Mass Spectrum Analysis; Methionine; Nitrogen; Parents; Peptide Hydrolases; Peptides; Phase; Plant Resins; Preparation; Promega; Research; Research Personnel; Resolution; Resources; Sampling; Source; Speed; TEP1 gene; Technology; Temperature; United States National Institutes of Health; Water; Width; ammonium bicarbonate; capillary; ionization; mass spectrometer; nano-electrospray; pressure

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. Sample preparation One hundred fifty micro grams of the 13C1-Ala FucT3 and 12C-AlaFucT3 were mixed and dried in speed vac. The mixed sample was dissolved in 100 ¿L of 50 mM ammonium bicarbonate buffer. The sample was immediately reduced with 25 mM dithiothreitol (45 min at 50oC) and carboxyamidomethylated with 90 mM iodoacetamide (45 min at room temperature in the dark). The sample was digested with sequence grade typsin (Promega, Madison, WI) overnight at 37¿C. After protease digestion, the sample was treated with 1% formic acid to deactivate the enzyme and then was passed through a C18 reversed phase cartridge. The sample was dried down in a speed vac. Analysis of peptides of TEP1 The peptides were resuspended in 39 ¿L of mobile phase A (0.1% formic acid) and 1 ¿L of mobile phase B (80% acetonitrile, in 0.1% formic acid) and then filtered with 0.2 ¿m filters (Nanosep, PALL). The sample was loaded by off- line onto a nanospray tapered capillary column/emitter (360 ¿ 75 ¿ 15 ¿m, PicoFrit, New Objective) self-packed with C18 reverse phase (RP) resin (10.5 cm, Waters) in a Nitrogen pressure bomb for 5 min at 1000 psi (~5 ¿L load) and then separated via a 160 min linear gradient of increasing mobile phase B at a flow rate of ~200 nL/min directly into the mass spectrometer. The separated peptides were analyzed by nanoelectrospray ionization mass spectrometry (NSI-MS) using an LTQ Orbitrap XL mass spectrometer (Thermofisher, San Jose, CA). A full FTMS (Fourier Transform Mass Spectrometry) spectrum at 60, 000 resolution was collected at m/z 300 to 2000 followed by 6 data dependent MS/MS spectra of ITMS (Ion Trap Mass Sepctrometry) in the most intense ion peaks following CID (36 % normalized collision energy). Dynamic mass increases of 15.9949 Da and 57.0215 Da were allowed from oxidized methionine and alkylated cystenine, respectively. The parent mass width was set up ¿ 20.0 ppm and reject mass list was applied to remove the contaminant peaks from column. The resulting data was searched against the FucT3 database using the TurboSequest algorithm. DTA files were generated for spectra with a threshold of 15 ions, a TIC of 1e3 and a range of [MH]+ 5005000 m/z. The SEQUEST parameters were set to allow ¿ 30.0 ppm of precursor ion mass tolerance and 0.5 Da of fragment ion tolerance with monoisotopic mass for LTQ Orbitrap XL data searching.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中央赠款提供的资源 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，在列出的机构中可能会被压制 对于中心，这不一定是调查员的工厂。 样品制备 将150个微语法混合在13c1-ala fuct3和12c-Alignuct3中。 样品在100中脱节。 l 50 mm碳酸氢铵缓冲液。 毫米二硫代硫醇（50oC时为45分钟），羧基固定剂，带90 mm碘乙酰胺（房间45分钟） 在黑暗中的温度）。 37¿ C.在特里特消化后，将样品用1％formic处理以停用酶，然后是 通过C18反向相位的墨盒。 TEP1肽的分析 将肽在39€中复活。 l流动阶段A（0.1％甲酸）和1» l流动期B（80％ 乙腈，在0.1％甲酸中），并用0.2€过滤M过滤器（Nanosep，PAL）。 一行在纳米喷的锥形毛细管柱/发射极（360€75？15€m，picofrit，新目标）上，与 C18反相（RP）树脂（10.5厘米，水域）在氮气中以1000 psi（〜5»l载荷）和 然后通过增加〜200 nl/min的流动相相位相相位相位的160分钟线性梯度分离 质谱仪。 使用LTQ通过纳米电喷雾质谱法（NSI-MS）分析分离的肽 Orbitrap XL质谱仪（Thermofisher，San Jose，CA）。 频谱在60时，分辨率以m/z 300至2000收集，然后是6个数据依赖的MS/MS光谱 （离子陷阱质量sepctrometry）在CID后最激烈的离子峰（36％归一化型型胶体能量） 从氧化的甲氧氨酸和烷基化的墨西甲胺中允许质量增加15.9949 DA和57.0215 DA， 分别设置了母体质量宽度� 20.0 ppm和拒绝质量列表被应用以消除污染物 圆柱的峰值。 使用TurboseQuest算法对FUCT3数据库进行搜索 用于15个离子的阈值，1E3的阈值和[MH]+ 5000 m/z的范围 将参数设置为允许� 30.0 ppm的前体离子质量耐受性和0.5 da的碎片离子耐受性， LTQ Orbitrap XL数据搜索的单异位素质量。