ANALYSIS OF ORGANELLES BY ON-LINE TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY-TANDEM M

在线二维液相色谱-TANDEM M分析细胞器

• 批准号: 7957738
• 负责人: JOHN L YATES
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957738
• 关键词: Amino Acid Sequence; Biology; Cation Exchange Resins; Charge; Computer Retrieval of Information on Scientific Projects Database; Coupled; Databases; Development; Dimensions; Funding; Fungal Genome; Grant; Hydrophobic Interactions; Institution; Liquid Chromatography; Mass Spectrum Analysis; Methods; Organelles; Organic solvent product; Peptides; Phase; Plant Resins; Protein Analysis; Proteins; Research; Research Personnel; Resources; Sodium Chloride; Source; System; United States National Institutes of Health; base; genome sequencing; tandem mass spectrometry; two-dimensional

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. The sequencing of the genomes of many different species has greatly helped our understanding of organelles. This information has driven the development of mass spectrometry (MS)-based methods for large-scale analysis of proteins. One of these methods uses two-dimensional liquid chromatography (2DLC) coupled on-line to tandem MS and is described here. In this method, proteins are first proteolytically digested, and then the peptides are separated in two dimensions. Typically, separation in the first dimension is based on charge interactions with a strong cation exchange (SCX) resin, whereas separation in the second dimension is based on hydrophobic interactions with a reversed-phase (RP) support. Peptides are eluted from the SCX resin using increasing concentrations of a salt and subsequently trapped on the RP resin. Next, the salt is washed from the system, and the peptides are eluted using an increasing gradient of a non-polar organic solvent. Eluting peptides are mass analyzed and fragmented to generate tandem mass spectra. These tandem mass spectra can be used to search sequence databases to identify peptides by matching amino-acid sequences to each spectrum.

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