High-performance MALDI-TOF-TOF for Proteomics

描述(申请人提供)：该项目的目标是开发一种新的高性能MALDI-TOF-TOF仪器，以消除限制蛋白质组学进展的主要瓶颈。人们普遍认为，串联质谱仪与高效分离相结合，才能达到对血浆、血清、尿液和脑脊液等生物体液进行全球分析所需的灵敏度和动态范围。对近端体液和组织样本的分析要求较低，因为所需的动态范围实质上较小，但可用的总样本量可能更有限，因此需要相同的最终灵敏度。多维分离结合质谱仪可以提供所需的动态范围，但需要大量的馏分，并且MS和MS-MS的测量必须非常快速和可靠，否则吞吐量不足以满足实际应用。已经开发或提出了几种分离方案，它们可以产生高达100,000个馏分/天，用于MS和MS-MS分析，平均每个馏分大约10-20个肽。已有的MS技术可以接近MS测量所需的速度，但目前的MS-MS技术都不在所需速度的数量级之内。 新的TOF-TOF将比目前的MS-MS仪器快几个数量级地产生高质量的MS-MS光谱。基本上，所有产生的光谱都将以相应的速度进行解释。通过使用比以前的MALDI仪器快25倍的激光速率和每次激光发射后的多个前体离子选择，实现了高速。该仪器的独特之处在于，它提供了MALDI MS-MS的高分辨率前体选择。可以选择单个同位素并将其碎裂到m/z 4000，离子传输没有可检测到的损失，相邻质量的贡献小于1%。该仪器还允许在MS-MS中进行高达50倍的多路传输。选定的质量必须至少相差1%，并且最好在强度的数量级范围内。这使得以前所未有的速度产生非常高质量的MS-MS光谱。出现在包含100个或更多峰的复杂多肽质量指纹中的所有多肽都可以在不耗尽样品的情况下进行碎片和鉴定。这使得MS-MS测量的速度和灵敏度与MS结果保持同步。将高分辨率前体选择与高激光速率和多路复用相结合，将允许在10 attomole/？L水平上对检测到的多肽产生高质量、可解释的MS-MS光谱。 随着人类基因组的完成，人们普遍认为“蛋白质组学是下一件大事”。不幸的是，这种乐观情绪催生的蛋白质组学工厂未能兑现承诺，已经倒闭。主要问题是动态范围不够。最丰富的蛋白质通常通过各种技术来检测，但丰度较低的蛋白质通常不会被检测到，特别是如果对确定的鉴定施加了严格的条件。通过对样品进行广泛的分馏，可以克服质谱学有限的动态范围，但这就要求必须分析非常大量的组分。新的飞行时间-飞行时间系统为这一问题提供了一个实用的解决方案。

DESCRIPTION (provided by applicant): The goal of the goal of this project is to develop a new high-performance MALDI-TOF-TOF instrument that removes a major bottleneck limiting progress in proteomics. It is widely recognized that a combination of tandem mass spectrometry with efficient separation is required to approach the sensitivity and dynamic range required for global analysis of biological fluids such as plasma, serum, urine, and CSF. Analysis of proximal fluids and tissue samples is somewhat less demanding in that the dynamic range required is substantially smaller, but the amount of total sample available may be more limited so that equal ultimate sensitivity is required. Multi-dimensional separation combined with mass spectrometry can provide the required dynamic range, but a large number of fractions are required, and the MS and MS-MS measurements must be very fast and reliable or throughput is insufficient for practical applications. Several separation schemes have been developed or proposed that may produce up to 100,000 fractions/day for MS and MS-MS analysis with ca. 10-20 peptides per fraction on average. MS techniques are available that can approach the speed required for the MS measurement, but no current MS-MS technique comes within an order of magnitude of the required speed. The new TOF-TOF will produce high-quality MS-MS spectra orders of magnitude faster than current MS-MS instruments. Essentially all of the spectra produced will be interpreted with corresponding speed. The high speed is achieved by using a laser rate 25 times faster than previous MALDI instruments and multiple precursor ion selection following each laser shot. This instrument is unique in that it provides high-resolution precursor selection with MALDI MS-MS. Single isotopes can be selected and fragmented up to m/z 4000 with no detectable loss in ion transmission and less than 1% contribution from adjacent masses. This instrument also allows up to 50 fold multiplexing in MS-MS. Selected masses must differ by at least 1%, and are preferably within an order of magnitude range in intensity. This allows the generation of very high quality MS-MS spectrum at unprecedented speed. All of the peptides present in a complex peptide mass fingerprint containing a hundred or more peaks can be fragmented and identified without exhausting the sample. This allows speed and sensitivity of the MS-MS measurements to keep pace with the MS results. The combination of high-resolution precursor selection with high laser rate and multiplexing will allow high-quality, interpretable MS-MS spectra to be generated on detected peptides at the 10 attomole/?L level. Following completion of the human genome it was widely felt that "proteomics was the next big thing." Unfortunately, the proteomics factories spawned by this euphoria have failed to deliver on their promises and have gone out of business. The major problem is that the dynamic range is inadequate. The most abundant proteins are routinely detected by a variety of techniques, but lower abundance proteins are usually not detected, particularly if stringent conditions for confident identification are imposed. The limited dynamic range of mass spectrometry can be overcome by extensive fractionation of the sample, but this then requires that very large numbers of fractions must be analyzed. The new TOF-TOF system provides a practical solution to this problem.