Parallel Peptide Tandem Mass Spectrometry (MS)

描述（由申请人提供）：这是5 R33 CA 099139 -04平行肽串联质谱法对程序NOT-OD-09-058 NIH宣布恢复法案资金可用于竞争性修订申请的修订申请。最初的R33奖项是围绕一种方法设计的，该方法通过片段化存在于宽（例如400-1400）m/z范围内的所有肽来增加蛋白质鉴定。该想法是这种鸟枪CID（或多重碰撞诱导解离）比标准鸟枪蛋白质组学方法更有效，所述标准鸟枪蛋白质组学方法在将样品引入串联质谱仪的HPLC期间使用dad依赖性离子选择。虽然这种方法有效，但效率被许多识别中的简并所抵消。这促使我们修改了基本的数据采集方法。我们进行了一项实验，以观察每个m/z“通道”中的所有肽，而不是在宽m/z范围内观察所有肽。显然，这需要更多的注射，但如下所述，该方法非常有效地增加了可信蛋白质鉴定的数量、蛋白质序列覆盖率和动态范围，所有这些都是在没有预先样品分级分离的情况下实现的。缺点是获取数据集所需的时间，通常需要连续五天的自动LC-MS/MS时间。在此，我们请求将R33延长一年，以优化我们称为独立于离子计数的肽采集（PACIFIC）的方法：目的1）优化独立于离子计数的肽采集（PACIFIC），使其定量和多路复用，并将采集时间减少到两天，目的2）使用前体离子独立的自顶向下算法（PIITA）通过修改湿和干实验室方法以及结合PACIFIC和目的3）来鉴定“小”蛋白质。识别和消除化学噪声源，在每个m/z通道上系统采集的PACIFIC数据集允许彻底了解所有可检测的肽/蛋白质（从低和高信号/噪声）以及如何减少非肽“噪声”源以帮助检测低丰度蛋白质。 公共卫生相关性：许多NIH项目的统一目标是大幅降低癌症死亡率。目前的想法是，这可以通过早期检测疾病标志物来实现;例如蛋白质和代谢物。初步数据支持我们的建议表明，新的蛋白质可以通过简单的修改，蛋白质组学数据的采集和分析的方式来确定。我们提出的计划将导致更容易的蛋白质发现方法，可以立即被实验室采用，寻找疾病的蛋白质标志物。

DESCRIPTION (provided by applicant): This is a revision application of 5R33CA099139-04 Parallel Peptide Tandem Mass Spectrometry to the program NOT-OD-09-058 NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The original R33 award was devised around a method to increase protein identifications by fragmenting all peptides present in a wide (e.g. 400-1400) m/z range. The idea being that this shotgun CID (or multiplexed collision induced dissociation is more efficient than standard shotgun proteomic methods that use dad-dependent ion selection during HPLC introduction of the sample to a tandem mass spectrometer. While this approach worked, the efficiency was offset by the degeneracy in many of the identifications. This led us to modify our basic data acquisition approach. Instead of looking at all peptides in a wide m/z range, we carried out an experiment to look at all peptides in each m/z "channel". Obviously, this requires many more injections, but as described below the approach worked remarkably well to increase the number of confident protein identifications, protein sequence coverage and dynamic range all of which was achieved without prior sample fractionation. The downside is the time it takes to acquire data set, typically five days of continuous automated LC-MS/MS time. Here we request a one year extension of our R33 to optimize a method we refer to as Peptide Acquisition Independent From Ion Count (PAcIFIC) by: Aim 1) Optimize Peptide Acquisition Independent From Ion Count (PAcIFIC) making it quantitative and multiplexed as well as reducing acquisition time to two days, Aim 2) Use Precursor Ion Independent Top- down Algorithm (PIITA) to identify "small" proteins by modification of wet and dry lab methods as well as to incorporate PAcIFIC and Aim 3) Identify and eliminate sources of chemical noise, PAcIFIC data sets being systematically acquired on each and every m/z channel allows a thorough understanding of all peptides/proteins detectable (from low and high signal/noise) and how sources of non-peptide "noise" may be diminished to aid detection of low abundance proteins. PUBLIC HEALTH RELEVANCE: A unifying goal of many NIH programs is to drastically reduce deaths from cancer. The current thinking is this may be achieved through early detection of markers of disease; e.g. proteins and metabolites. Preliminary data supporting our proposal shows that new proteins may be identified by simple modifications to the way proteomic data is acquired and analyzed. Our proposed plan will lead to more facile methods for protein discovery that may be immediately adopted by laboratories searching for protein markers of disease.