A NOVEL DUAL-PRESSURE LINEAR ION TRAP IMPROVES THE ANALYSIS OF PROTEOMICS SAMPLE

新型双压线性离子阱改善蛋白质组样品的分析

• 批准号: 8171217
• 负责人: Michael MacCoss
• 金额: $ 0.67万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171217
• 关键词: Caenorhabditis elegans; Complex; Computer Retrieval of Information on Scientific Projects Database; Detection; Funding; Grant; Institution; Ions; Myoglobin; Penetration; Peptides; Proteins; Proteome; Proteomics; Research; Research Personnel; Resolution; Resources; Saccharomyces cerevisiae; Sampling; Source; Time; United States National Institutes of Health; base; design; improved; instrument; instrumentation; mass spectrometer; novel; pressure; protein aminoacid sequence; research study; tandem mass spectrometry

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 dramatic progress in high throughput proteomics analysis via ESI-LC/MS over the last decade has been fueled to a large degree by continuous improvements in instrumentation. High throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become the dominant analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ VelosTM) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times two times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. Faster cycle times on the new instrument alternatively allow for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. The greatly increased ion flux into the mass spectrometer also results in greatly increased detection of low abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker?s yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Additionally, mass analysis in the new instrument can be carried out with resolution in excess of 20,000 FWHM, sufficient for isotopic resolution and top down experiments of small intact proteins, such as myoglobin.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在过去的十年中，通过ESI-LC/MS的高通量蛋白质组学分析的巨大进步在很大程度上是由仪器的不断改进推动的。 高通量鉴定实验基于肽测序，并且主要通过使用串联质谱来完成，其中离子阱和基于阱的仪器已经成为主要的分析平台。为了满足对表征深度和吞吐量日益苛刻的要求，我们提出了一种新开发的双压线性离子阱质谱仪（LTQ VelosTM），该质谱仪具有更高的灵敏度，通过新的源设计提供，并展示了比LTQ XL短两倍的实际循环时间，同时提高或保持MS/MS裂解光谱的光谱质量。 与现有平台相比，这些改进导致从秀丽隐杆线虫复杂蛋白质组中检测和鉴定蛋白质和独特肽的大幅增加。 新仪器上更快的循环时间也允许在给定深度的蛋白质组分析中实现更高的通量，使用LTQ Velos在60分钟内识别的肽和蛋白质比使用LTQ XL在180分钟内识别的肽和蛋白质更多。 进入质谱仪的大大增加的离子通量也导致大大增加的低丰度肽的检测。 这些改进累积起来导致了更大的渗透到面包师？s酵母（酿酒酵母）蛋白质组相比，LTQ XL。 此外，新仪器中的质量分析可以以超过20，000 FWHM的分辨率进行，足以进行同位素分辨率和小型完整蛋白质（如肌红蛋白）的自上而下实验。