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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在过去的十年里，通过ESI-LC/MS在高通量蛋白质组学分析方面取得了巨大的进步，这在很大程度上是由仪器的不断改进推动的。高通量鉴定实验是以肽序列为基础的，主要通过使用串联质谱仪完成，离子陷阱和基于陷阱的仪器已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力线性离子陷阱质谱仪(LTQ VelosTM)，其特点是通过新的源设计提高了灵敏度，并证明了实际周期比LTQ XL短两倍，同时改善或保持了MS/MS碎裂光谱的光谱质量。与现有平台相比，这些改进大大增加了对秀丽线虫复杂蛋白质组中蛋白质和独特多肽的检测和鉴定。新仪器的循环时间更快，或者允许在给定深度的蛋白质组分析中获得更高的吞吐量，使用LTQ Velos在60分钟内识别出比使用LTQ XL在180分钟内识别出的更多多肽和蛋白质。进入质谱仪的离子通量大大增加，也大大增加了对低丰度多肽的检测。与LTQ XL相比，这些改进累积起来导致了面包师S酵母(酿酒酵母)蛋白质组的渗透率大大提高。此外，新仪器中的质量分析可以以超过20,000个半高宽的分辨率进行，足以进行同位素分辨率和对小的完整蛋白质(如肌红蛋白)的自上而下的实验。

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.