High-performance MALDI-TOF-TOF for Proteomics

描述（由申请人提供）：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，串联质谱与高效分离的结合需要接近生物流体（如血浆、血清、尿液和脑脊液）全局分析所需的灵敏度和动态范围。近端液体和组织样本的分析要求较低，因为所需的动态范围要小得多，但可用的总样本数量可能更有限，因此需要相同的最终灵敏度。多维分离结合质谱法可以提供所需的动态范围，但需要大量的组分，而且MS和MS-MS测量必须非常快速和可靠，否则通量不足以用于实际应用。已经开发或提出了几种分离方案，可以产生多达100,000个馏分/天用于MS和MS-MS分析，平均每个馏分约10-20个肽。可用的质谱技术可以接近质谱测量所需的速度，但目前的质谱技术没有达到所需速度的数量级。

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.