Advancing Multiplexed Isobaric Tag-based Strategies for Proteome Profiling

摘要 样品多路传输是最近许多大规模蛋白质组学倡议的催化剂。……的到来 等压标记，如iTRAQ(相对和绝对定量的等压标记)和TMT所普及的 (串联质量标签)试剂，已成为多重蛋白质表达的典型方法 侧写。存在两种主要的数据采集方法，每种方法都有其优缺点。首先， 仅MS2方法(这里称为MS2-IDQ)可以在单个光谱中鉴定(ID)和定量(Q)一个肽。 第二，同步前体选择(SPS)-MS3方法在MS2阶段识别前体，但是 然后从MS2阶段选择一系列碎片离子，这些碎片离子被进一步碎裂并读出为MS3 用于量化测量的光谱。MS2-IDQ遭受共同隔离和共同碎片化的困扰 前体离子(“干扰”)，虽然SPS-MS3有助于减轻干扰，但这是以牺牲 速度，这是获取长MS3扫描的直接结果。在这里，我们旨在开发、评估和应用 新的数据采集平台，融合了当前方法的优点，并缓解了它们的主要警告。 ThermoFisher Science的Orbitrap Fusion和Lumos仪器的最新进展是 实施允许扩展控制的仪器应用程序编程接口(IAPI) 该仪器超出了制造商的内置功能。使用此界面，Gygi实验室和 其他公司已经开始创建定制的实时搜索(RTS)算法。RTS支持MS2 实时搜索频谱，并决定MS3扫描是否可能导致 一项重要的多肽量化测量。通过省略MS3扫描，可以收集更多的MS2频谱 新的多肽可能会被识别出来。使用iAPI，可以添加函数，包括目标列表和限制 设置为每个蛋白质(在非常丰富和/或大的蛋白质的情况下)定量的多肽数量，这 在翻译研究中很有用，例如对血浆样本和其他体液的询问。 我们的具体目标是进一步发展成功应用RTS的方法- MS3.在具体目标1中，我们将使用TKO和TKO对RTS-MS3的新兴算法进行基准测试 基于TMT的蛋白质组谱分析的超级(人-酵母多肽资源)标准。在具体目标2中，我们 将在几种样本类型(细菌培养、小鼠组织、血液、 脑脊液、人类细胞系和酵母菌培养)与传统的MS2-IDQ和SPS-MS3方法不同 (具体目标2)。最后，在具体目标3中，我们将应用RTS-MS3平台对整个酵母进行分析 在两种生长条件下收集缺失菌株，这将产生最大的酵母蛋白表达 分析到目前为止的数据集。实现这三个具体目标将使RTS-MS3平台成为 颠覆性技术对当前基于等压标签的多路复用方法的影响，并将标志着 基于等压标签的定量蛋白质组学。

ABSTRACT Sample multiplexing has been the catalyst for many recent large-scale proteomics initiatives. The advent of isobaric tagging, as popularized by iTRAQ (isobaric tags for relative and absolute quantitation) and TMT (tandem mass tag) reagents, has become the quintessential methodology for multiplexed protein expression profiling. Two major data acquisition methods exist each with its own advantages and disadvantages. First, the MS2-only method (“MS2-IDQ” herein) can identify (ID) and quantify (Q) a peptide in a single spectrum. Second, the synchronous precursor selection (SPS)-MS3 method identifies the precursor in the MS2 stage, but then selects a series of fragment ions from the MS2 stage that are fragmented further and read out as an MS3 spectrum for quantification measurements. MS2-IDQ suffers from the co-isolation and co-fragmentation of precursor ions (“interference”), and although SPS-MS3 helps to alleviate interference, it is at the expense of speed, a direct result from the acquisition of long MS3 scans. Here we aim to develop, evaluate, and apply a novel data acquisition platform that merges the benefits of current methods and alleviates their major caveats. A recent development on ThermoFisher Scientific's Orbitrap Fusion and Lumos instruments has been the implementation of an instrument application programming interface (iAPI) that allows for expanded control of the instrumentation beyond the manufacturer's built-in functionality. Using this interface, the Gygi Lab and others have begun to create custom on-the-fly real-time search (RTS) algorithms. RTS enables an MS2 spectrum to be searched in real-time and decisions to be made as to whether an MS3 scan is likely to result in a significant peptide quantification measurement. By omitting MS3 scans, more MS2 spectra can be collected and new peptides may be identified. Using the iAPI, functions can be added including targeted lists and limits set for the number of peptides quantified per protein (in the case of very abundant and/or large proteins), which is useful in translational research, such as the interrogation of plasma samples and other body fluids. Our Specific Aims are geared toward developing further the methodology for successful application of RTS- MS3. In Specific Aim 1, we will benchmark emerging algorithms for RTS-MS3 using both the TKO and HYPER (human-yeast peptide resource) standards for TMT-based proteome profiling. In Specific Aim 2, we will evaluate the RTS-MS3 platform across several sample types (bacterial cultures, mouse tissues, blood, cerebral spinal fluid, human cell lines, and yeast cultures) against traditional MS2-IDQ and SPS-MS3 methods (Specific Aim 2). Finally, in Specific Aim 3 we will apply the RTS-MS3 platform to analyze an entire Yeast Deletion Strain Collection under two growth conditions, which will produce the largest yeast protein expression profiling data set to date. Accomplishing these three Specific Aims will establish the RTS-MS3 platform as a disruptive technology to current isobaric tag-based multiplexing methodology and will mark a paradigm shift in isobaric tag-based quantitative proteomics.